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Sample records for primary cultured normal

  1. Lipid-mediated transfection of normal adult human hepatocytes in primary culture.

    PubMed

    Ourlin, J C; Vilarem, M J; Daujat, M; Harricane, M C; Domergue, J; Joyeux, H; Baulieux, J; Maurel, P

    1997-04-05

    The aim of this work was to develop a procedure for the lipid-mediated transfection of DNA into normal adult human hepatocytes in culture. Cells were plated in a serum-free culture medium at various cell densities, on plastic or collagen-coated dishes, both in the absence and in the presence of epidermal growth factor (EGF). The cells were incubated for various periods of time with mixtures of DNA-lipofectin or DNA-3 beta[N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol (DC-chol) liposomes, and the efficiency of transfection was assessed by measuring the activity of reporter genes, beta-galactosidase or chloramphenicol acetyl-transferase (CAT). For comparison, similar experiments were carried out with human cell lines including HepG2, Caco-2, and WRL68. The efficiency of transfection (in percentage of cells) was not significantly different after transfection with lipofectin or DC-chol and comprised between 0.04 and 1.7% (extreme values) for different cultures. The efficiency of transfection decreased as the age or density of the culture increased and increased in cultures treated with EGF. Direct measurement of the rate of DNA synthesis suggested that the efficiency of transfection was related to the number of cells entering the S phase. Under the same conditions, the efficiency of transfection was one to two orders of magnitude greater in the three cell lines. A plasmid harboring 660 bp of the 5'-flanking region of CYP1A1 (containing two xenobiotic enhancer elements) fused upstream of the promoter of thymidine kinase and the CAT reporter gene was constructed. When this plasmid was transfected in human hepatocytes, CAT activity was induced as expected. We conclude that normal adult human hepatocytes can be transfected with exogenous DNA and that the transfected construct is regulated in the manner expected from in vivo studies.

  2. Magnetic Nanodrug Delivery Through the Mucus Layer of Air-Liquid Interface Cultured Primary Normal Human Tracheobronchial Epithelial Cells.

    PubMed

    Economou, E C; Marinelli, S; Smith, M C; Routt, A A; Kravets, V V; Chu, H W; Spendier, K; Celinski, Z J

    2016-09-01

    Superparamagnetic iron oxide (Fe3O4) and highly anisotropic barium hexaferrite (BaFe12O19) nanoparticles were coated with an anti-inflammatory drug and magnetically transported through mucus produced by primary human airway epithelial cells. Using wet planetary ball milling, dl-2-amino-3-phosphonopropionic acid-coated BaFe12O19 nano-particles (BaNPs) of 1-100 nm in diameter were prepared in water. BaNPs and conventional 20-30-nm Fe3O4 nanoparticles (FeNPs) were then encased in a polymer (PLGA) loaded with dexamethasone (Dex) and tagged for imaging. PLGA-Dex-coated BaNPs and FeNPs were characterized using dynamic light scattering (DLS), transmission electron microscopy (TEM), and superconducting quantum interference device (SQUID) magnetometry. Both PLGA-Dex-coated BaNPs and FeNPs were transferred to the surface of a ~100-μm thick mucus layer of air-liquid interface cultured primary normal human tracheobronchial epithelial (NHTE) cells. Within 30 min, the nanoparticles were pulled successfully through the mucus layer by a permanent neodymium magnet. The penetration time of the nanomedicine was monitored using confocal microscopy and tailored by varying the thickness of the PLGA-Dex coating around the particles.

  3. Magnetic Nanodrug Delivery Through the Mucus Layer of Air-Liquid Interface Cultured Primary Normal Human Tracheobronchial Epithelial Cells

    PubMed Central

    Economou, E. C.; Marinelli, S.; Smith, M. C.; Routt, A. A.; Kravets, V. V.; Chu, H. W.; Spendier, K.; Celinski, Z. J.

    2016-01-01

    Superparamagnetic iron oxide (Fe3O4) and highly anisotropic barium hexaferrite (BaFe12O19) nanoparticles were coated with an anti-inflammatory drug and magnetically transported through mucus produced by primary human airway epithelial cells. Using wet planetary ball milling, dl-2-amino-3-phosphonopropionic acid-coated BaFe12O19 nano-particles (BaNPs) of 1–100 nm in diameter were prepared in water. BaNPs and conventional 20–30-nm Fe3O4 nanoparticles (FeNPs) were then encased in a polymer (PLGA) loaded with dexamethasone (Dex) and tagged for imaging. PLGA-Dex-coated BaNPs and FeNPs were characterized using dynamic light scattering (DLS), transmission electron microscopy (TEM), and superconducting quantum interference device (SQUID) magnetometry. Both PLGA-Dex-coated BaNPs and FeNPs were transferred to the surface of a ~100-μm thick mucus layer of air-liquid interface cultured primary normal human tracheobronchial epithelial (NHTE) cells. Within 30 min, the nanoparticles were pulled successfully through the mucus layer by a permanent neodymium magnet. The penetration time of the nanomedicine was monitored using confocal microscopy and tailored by varying the thickness of the PLGA-Dex coating around the particles. PMID:27774374

  4. Cultured normal mammalian tissue and process

    NASA Technical Reports Server (NTRS)

    Goodwin, Thomas J. (Inventor); Prewett, Tacey L. (Inventor); Wolf, David A. (Inventor); Spaulding, Glenn F. (Inventor)

    1993-01-01

    Normal mammalian tissue and the culturing process has been developed for the three groups of organ, structural and blood tissue. The cells are grown in vitro under microgravity culture conditions and form three dimensional cell aggregates with normal cell function. The microgravity culture conditions may be microgravity or simulated microgravity created in a horizontal rotating wall culture vessel.

  5. Effects of insulin, dexamethasone and cytokines on {alpha}{sub 1}-acid glycoprotein gene expression in primary cultures of normal rat hepatocytes

    SciTech Connect

    Barraud, B.; Balavoine, S.; Feldmann, G.; Lardeux, B.

    1996-04-01

    While the effects of insulin, dexamethasone and cytokines on {alpha}{sub 1}-acid glycoprotein gene expression have been investigated in various hepatoma cell lines, the individual and combined effects of these components on the expression of this gene have been rarely studied in cultured normal rat hepatocytes. In this cell model, we have shown that mRNA levels of {alpha}{sub 1}-acid glycoprotein were not decreased at least during the first 24 h of culture under basal conditions. During these short-term cultures, the expression of {alpha}{sub 1}-acid glycoprotein in normal hepatocytes showed a high degree of responsiveness to dexamethasone alone (20-fold increase) and to dexamethasone associated with various cytokines (interleukin-1{beta}, interleukin-6 and tumor necrosis factor {alpha}) with a 40 to 100-fold increase depending on the cytokine. Insulin alone did not modify {alpha}{sub 1}-acid glycoprotein mRNA; however, this hormone exerted a positive effect (about 50% increase) in the presence of dexamethasone or dexamethasone with cytokines. These results indicate that the regulation of {alpha}{sub 1}-acid glycoprotein in cultured normal rat hepatocytes presents major differences when compared to reported observations in rat hepatoma cell lines. 49 refs., 2 figs., 2 tabs.

  6. Three Dimensional Primary Hepatocyte Culture

    NASA Technical Reports Server (NTRS)

    Yoffe, Boris

    1998-01-01

    Our results demonstrated for the first time the feasibility of culturing PHH in microgravity bioreactors that exceeded the longest period obtained using other methods. Within the first week of culture, isolated hepatocytes started to form aggregates, which continuously increased in size (up to 1 cm) and macroscopically appeared as a multidimensional tissue-like assembly. To improve oxygenation and nutrition within the spheroids we performed experiments with the biodegradable nonwoven fiber-based polymers made from PolyGlycolic Acid (PGA). It has been shown that PGA scaffolds stimulate isolated cells to regenerate tissue with defined sizes and shapes and are currently being studied for various tissue-engineering applications. Our data demonstrated that culturing hepatocytes in the presence of PGA scaffolds resulted in more efficient cell assembly and formations of larger cell spheroids (up to 3 cm in length, see figure). The histology of cell aggregates cultured with PGA showed polymer fibers with attached hepatocytes. We initiated experiments to co-culture primary human hepatocytes with human microvascular endothelial cells in the bioreactor. The presence of endothelial cells in co-cultures were established by immunohistochemistry using anti-CD34 monoclonal Ab. Our preliminary data demonstrated that cultures of purified hepatocytes with human microvascular endothelial cells exhibited better growth and expressed higher levels of albumin MRNA for a longer period of time than cultures of ppfified, primary human hepatocytes cultured alone. We also evaluated microsomal deethylation activity of hepatocytes cultured in the presence of endothelial cells.In summary, we have established liver cell culture, which mimicked the structure and function of the parent tissue.

  7. Kinetics and regulation of human keratinocyte stem cell growth in short-term primary ex vivo culture. Cooperative growth factors from psoriatic lesional T lymphocytes stimulate proliferation among psoriatic uninvolved, but not normal, stem keratinocytes.

    PubMed Central

    Bata-Csorgo, Z; Hammerberg, C; Voorhees, J J; Cooper, K D

    1995-01-01

    Flow cytometric analysis of primary ex vivo keratinocyte cultures demonstrated that stem cells, (beta 1 integrin+, keratin 1/keratin 10 [K1/K10-], proliferating cell nuclear antigen [PCNA-] [Bata-Csorgo, Zs., C. Hammerberg, J. J. Voorhees, and K. D. Cooper. 1993. J. Exp. Med. 178:1271-1281]) establish such cultures. This methodology also enabled the quantitation of synchronized recruitment of these cells from G0 into G1 of the cell cycle (PCNA expression), which preceded bright beta 1 integrin expression. (beta 1 integrinbright expression has been shown to be a characteristic feature of keratinocyte stem cells in culture (Jones, P. H., and F. M. Watt. 1993. Cell. 73:713-724). Using the above assay, we determined whether lesional T lymphocytes in psoriasis could be directly responsible for the induction of the stem cell hyperproliferation that is characteristic of this disease. Indeed, CD4+ T lymphocytes, cloned from lesional psoriatic skin and stimulated by immobilized anti-CD3 plus fibronectin, promoted psoriatic uninvolved keratinocyte stem cell proliferation via soluble factors. This induction appeared to be through accelerated recruitment of stem cells from their quiescent state (G0) into cell cycle. By contrast, normal keratinocyte stem cells exhibited no such growth stimulation. Supernatants exhibiting growth induction all contained high levels of GM-CSF and gamma-IFN, low IL-3 and TNF-alpha, and variable IL-4. Only anti-gamma-IFN antibody was able to neutralize growth stimulatory activity of the supernatants on psoriatic uninvolved keratinocyte stem cells. However, because recombinant gamma-IFN alone inhibited growth in this assay, these data suggest that, in psoriasis, gamma-IFN acts cooperatively with other growth factors in the immune induction of cell cycle progression by the normally quiescent stem cell keratinocytes. Images PMID:7529261

  8. The current status of primary hepatocyte culture

    PubMed Central

    Mitaka, Toshihiro

    1998-01-01

    Recently, there have been significant advances toward the development of culture conditions that promote proliferation of primary rodent hepatocytes. There are two major methods for the multiplication of hepatocytes in vitro: one is the use of nicotinamide, the other is the use of a nutrient-rich medium. In the medium containing a high concentration of nicotinamide and a growth factor, primary hepatocytes can proliferate well. In this culture condition small mononucleate cells, which are named small hepatocytes, appear and form colonies. Small hepatocytes have a high potential to proliferate while maintaining hepatic characteristics, and can differentiate into mature ones. On the other hand, combining the nutrient-rich medium with 2% DMSO, the proliferated hepatocytes can recover the hepatic differentiated functions and maintain them for a long time. In this review I describe the culture conditions for the proliferation and differentiation of primary hepatocytes and discuss the small hepatocytes, especially their roles in liver growth. PMID:10319020

  9. PRIMARY CULTURES OF DISSOCIATED SYMPATHETIC NEURONS

    PubMed Central

    Mains, Richard E.; Patterson, Paul H.

    1973-01-01

    Rat sympathetic ganglia were disrupted by mechanical agitation to yield dissociated primary neurons, and the conditions for long-term growth in culture of the isolated neurons were examined. The neurons were grown with or without non-neural cells, simply by the addition or deletion of bicarbonate during growth in culture. Fluorescence histochemistry indicated that the isolated neurons contained catecholamines; incubations with radioactive precursors were used to verify the synthesis and accumulation of both dopamine and norepinephrine. The neurons also produced octopamine using tyramine as precursor, but not with tyrosine as the precursor. In the presence of eserine, older cultures synthesized and stored small amounts of acetylcholine. The cultures did not synthesize and accumulate detectable levels of radioactive γ-aminobutyric acid, 5-hydroxytryptamine, or histamine. PMID:4616046

  10. Dependability of sensitivity tests in primary culture.

    PubMed Central

    Waterworth, P M; Del Piano, M

    1976-01-01

    Primary sensitivity tests were done on 90 specimens of infected urine, and the results were compared with those of secondary tests on pure cultures done by three diffusion methods. There was good correlation between the four methods. In a second study, the reliability of primary tests prepared in the clinical laboratory on specimens of pus was assessed, and the frequency with which a definitive result was obtained with different types of specimen was determined. Recommendations are made for the economic use of these tests. Images PMID:1270599

  11. Molluscan cells in culture: primary cell cultures and cell lines

    PubMed Central

    Yoshino, T. P.; Bickham, U.; Bayne, C. J.

    2013-01-01

    In vitro cell culture systems from molluscs have significantly contributed to our basic understanding of complex physiological processes occurring within or between tissue-specific cells, yielding information unattainable using intact animal models. In vitro cultures of neuronal cells from gastropods show how simplified cell models can inform our understanding of complex networks in intact organisms. Primary cell cultures from marine and freshwater bivalve and gastropod species are used as biomonitors for environmental contaminants, as models for gene transfer technologies, and for studies of innate immunity and neoplastic disease. Despite efforts to isolate proliferative cell lines from molluscs, the snail Biomphalaria glabrata Say, 1818 embryonic (Bge) cell line is the only existing cell line originating from any molluscan species. Taking an organ systems approach, this review summarizes efforts to establish molluscan cell cultures and describes the varied applications of primary cell cultures in research. Because of the unique status of the Bge cell line, an account is presented of the establishment of this cell line, and of how these cells have contributed to our understanding of snail host-parasite interactions. Finally, we detail the difficulties commonly encountered in efforts to establish cell lines from molluscs and discuss how these difficulties might be overcome. PMID:24198436

  12. In vitro methods to culture primary human breast epithelial cells.

    PubMed

    Raouf, Afshin; Sun, Yu Jia

    2013-01-01

    Current evidence suggests that much like leukemia, breast tumors are maintained by a small subpopulation of tumor cells that have stem cell properties. These cancer stem cells are envisaged to be responsible for tumor formation and relapse. Therefore, knowledge about their nature will provide a platform to develop therapies to eliminate these breast cancer stem cells. This concept highlights the need to understand the mechanisms that regulate the normal functions of the breast stem cells and their immediate progeny as alterations to these same mechanisms can cause these primitive cells to act as cancer stem cells. The study of the primitive cell functions relies on the ability to isolate them from primary sources of breast tissue. This chapter describes processing of discarded tissue from reduction mammoplasty samples as sources of normal primary human breast epithelial cells and describes cell culture systems to grow single-cell suspensions prepared from these reduction samples in vitro.

  13. Reference gene for primary culture of prostate cancer cells.

    PubMed

    Souza, Aline Francielle Damo; Brum, Ilma Simoni; Neto, Brasil Silva; Berger, Milton; Branchini, Gisele

    2013-04-01

    Selection of reference genes to normalize mRNA levels between samples is critical for gene expression studies because their expression can vary depending on the tissues or cells used and the experimental conditions. We performed ten cell cultures from samples of prostate cancer. Cells were divided into three groups: control (with no transfection protocol), cells transfected with siRNA specific to knockdown the androgen receptor and cells transfected with inespecific siRNAs. After 24 h, mRNA was extracted and gene expression was analyzed by Real-time qPCR. Nine candidates to reference genes for gene expression studies in this model were analyzed (aminolevulinate, delta-, synthase 1 (ALAS1); beta-actin (ACTB); beta-2-microglobulin (B2M); glyceraldehyde-3-phosphate dehydrogenase (GAPDH); hypoxanthine phosphoribosyltransferase 1 (HPRT1); succinate dehydrogenase complex, subunit A, flavoprotein (Fp) (SDHA); TATA box binding protein (TBP); ubiquitin C (UBC); tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide (YWHAZ)). Expression stability was calculated NormFinder algorithm to find the most stable genes. NormFinder calculated SDHA as the most stable gene and the gene with the lowest intergroup and intragroup variation, and indicated GAPDH and SDHA as the best combination of two genes for the purpose of normalization. Androgen receptor mRNA expression was evaluated after normalization by each candidate gene and showed statistical difference in the transfected group compared to control group only when normalized by combination of GAPDH and SDHA. Based on the algorithm analysis, the combination of SDHA and GAPDH should be used to normalize target genes mRNA levels in primary culture of prostate cancer cells submitted to transfection with siRNAs.

  14. Predicting Organizational Commitment from Organizational Culture in Turkish Primary Schools

    ERIC Educational Resources Information Center

    Ipek, Cemalettin

    2010-01-01

    This study aims to describe organizational culture and commitment and to predict organizational commitment from organizational culture in Turkish primary schools. Organizational Culture Scale (Ipek "1999") and Organizational Commitment Scale (Balay "2000") were used in the data gathering process. The data were collected from…

  15. Examining School Culture in Flemish and Chinese Primary Schools

    ERIC Educational Resources Information Center

    Zhu, Chang; Devos, Geert; Tondeur, Jo

    2014-01-01

    The aim of this research is to gain understanding about school culture characteristics of primary schools in the Flemish and Chinese context. The study was carried out in Flanders (Belgium) and China, involving a total of 44 Flemish schools and 40 Chinese schools. The School Culture Scales were used to measure five school culture dimensions with…

  16. Teaching Cultural History from Primary Events

    ERIC Educational Resources Information Center

    Carson, Robert N.

    2004-01-01

    This article explores the relationship between specific cultural events such as Galileo's work with the pendulum and a curriculum design that seeks to establish in skeletal form a comprehensive epic narrative about the co-evolution of cultural systems and human consciousness. The article explores some of the challenges and some of the strategies…

  17. CHANGES IN GENE EXPRESSION DURING DIFFERENTIATION OF CULTURED HUMAN PRIMARY BRONCHIAL EPITHELIAL CELLS

    EPA Science Inventory

    Primary airway epithelial cell cultures are a useful tool for the in vitro study of normal bronchial cell differentiation and function, airway disease mechanisms, and pathogens and toxin response. Growth of these cells at an air-liquid interface for several days results in the f...

  18. Grounding Signs of Culture: Primary Intersubjectivity in Social Semiosis

    ERIC Educational Resources Information Center

    Cowley, Stephen J.; Moodley, Sheshni; Fiori-Cowley, Agnese

    2004-01-01

    The article examines how infants are first permeated by culture. Building on Thibault (2000), semiogenesis is traced to the joint activity of primary intersubjectivity. Using an African example, analysis shows how--at 14 weeks--an infant already uses culturally specific indicators of "what a caregiver wants." Human predispositions and…

  19. Mapping the cellular and molecular heterogeneity of normal and malignant breast tissues and cultured cell lines

    PubMed Central

    2010-01-01

    Introduction Normal and neoplastic breast tissues are comprised of heterogeneous populations of epithelial cells exhibiting various degrees of maturation and differentiation. While cultured cell lines have been derived from both normal and malignant tissues, it remains unclear to what extent they retain similar levels of differentiation and heterogeneity as that found within breast tissues. Methods We used 12 reduction mammoplasty tissues, 15 primary breast cancer tissues, and 20 human breast epithelial cell lines (16 cancer lines, 4 normal lines) to perform flow cytometry for CD44, CD24, epithelial cell adhesion molecule (EpCAM), and CD49f expression, as well as immunohistochemistry, and in vivo tumor xenograft formation studies to extensively analyze the molecular and cellular characteristics of breast epithelial cell lineages. Results Human breast tissues contain four distinguishable epithelial differentiation states (two luminal phenotypes and two basal phenotypes) that differ on the basis of CD24, EpCAM and CD49f expression. Primary human breast cancer tissues also contain these four cellular states, but in altered proportions compared to normal tissues. In contrast, cultured cancer cell lines are enriched for rare basal and mesenchymal epithelial phenotypes, which are normally present in small numbers within human tissues. Similarly, cultured normal human mammary epithelial cell lines are enriched for rare basal and mesenchymal phenotypes that represent a minor fraction of cells within reduction mammoplasty tissues. Furthermore, although normal human mammary epithelial cell lines exhibit features of bi-potent progenitor cells they are unable to differentiate into mature luminal breast epithelial cells under standard culture conditions. Conclusions As a group breast cancer cell lines represent the heterogeneity of human breast tumors, but individually they exhibit increased lineage-restricted profiles that fall short of truly representing the intratumoral

  20. Methylmalonate toxicity in primary neuronal cultures.

    PubMed

    McLaughlin, B A; Nelson, D; Silver, I A; Erecinska, M; Chesselet, M F

    1998-09-01

    Several inhibitors of mitochondrial complex II cause neuronal death in vivo and in vitro. The goal of the present work was to characterize in vitro the effects of malonate (a competitive blocker of the complex) which induces neuronal death in a pattern similar to that seen in striatum in Huntington's disease. Exposure of striatal and cortical cultures from embryonic rat brain for 24 h to methylmalonate, a compound which produces malonate intracellularly, led to a dose-dependent cell death. Methylmalonate (10 mM) caused >90% mortality of neurons although cortical cells were unexpectedly more vulnerable. Cell death was attenuated in a medium containing antioxidants. Further characterization revealed that DNA laddering could be detected after 3 h of treatment. Morphological observations (videomicroscopy and Hoechst staining) showed that both necrotic and apoptotic cell death occurred in parallel; apoptosis was more prevalent. A decrease in the ATP/ADP ratio was observed after 3 h of treatment with 10 mM methylmalonate. In striatal cultures it occurred concomitantly with a decline in GABA and a rise in aspartate content and the aspartate/glutamate ratio. Changes in ion concentrations were measured in similar cortical cultures from mouse brain. Neuronal [Na+]i increased while [K+]i and membrane potential decreased after 20 min of continuous incubation in 10 mM methylmalonate. These changes progressed with time, and a rise in [Ca2+]i was also observed after 1 h. The results demonstrate that malonate collapses cellular ion gradients, restoration of which imposes an additional load on the already compromised ATP-generation machinery. An early elevation in [Ca2+]i may trigger an increase in activity of proteases, lipases and endonucleases and production of free radicals and DNA damage which, ultimately, leads to cells death. The data also suggest that maturational and/or extrinsic factors are likely to be critical for the increased vulnerability of striatal neurons to

  1. Increased phase synchronization of spontaneous calcium oscillations in epileptic human versus normal rat astrocyte cultures

    NASA Astrophysics Data System (ADS)

    Balázsi, Gábor; Cornell-Bell, Ann H.; Moss, Frank

    2003-06-01

    Stochastic synchronization analysis is applied to intracellular calcium oscillations in astrocyte cultures prepared from epileptic human temporal lobe. The same methods are applied to astrocyte cultures prepared from normal rat hippocampus. Our results indicate that phase-repulsive coupling in epileptic human astrocyte cultures is stronger, leading to an increased synchronization in epileptic human compared to normal rat astrocyte cultures.

  2. Primary cell culture of human adenocarcinomas--practical considerations.

    PubMed

    Lerescu, Lucian; Tucureanu, Cătălin; Caraş, Iuliana; Neagu, Stefan; Melinceanu, Laura; Sălăgeanu, Aurora

    2008-01-01

    Cell culture is one of the major tools for oncology research, being an excellent system in which to study the biochemistry and molecular biology associated with individual cancer types and to understand cancer cell physiology. Progress in understanding the biology of any type of carcinoma has been impeded by the inability to culture adequately malignant cells from most epithelial tissues. The ultimate in vitro tumor model would completely reflect the in vivo tumor microenvironment in function and mechanism. Unfortunately, such a model does not currently exist. Homogeneous cell lines that can be continuously propagated on plastic surfaces have been extensively used as a surrogate for tumor environment; however they are very different from the in vivo tumor cells. Model systems involving primary culture represent the situation most closely related to the original tissue although they have a number of disadvantages over cell lines, such as the limited ability to repeat studies with a well characterized culture system that can be used in multiple laboratories. The primary culture may contain many types of stromal and infiltrating cell types potentially complicating the interpretation of data. Yet, their properties better reflect the cellular interactions present in intact tissue. The present article reviews the critical steps in obtaining, routine maintenance and cryopreservation of primary tumor cell cultures, based on information from literature and personal experience on the subject. The article also includes an updated protocol for primary tumor cell isolation and culture.

  3. Relevant principal factors affecting the reproducibility of insect primary culture.

    PubMed

    Ogata, Norichika; Iwabuchi, Kikuo

    2017-02-22

    The primary culture of insect cells often suffers from problems with poor reproducibility in the quality of the final cell preparations. The cellular composition of the explants (cell number and cell types), surgical methods (surgical duration and surgical isolation), and physiological and genetic differences between donors may be critical factors affecting the reproducibility of culture. However, little is known about where biological variation (interindividual differences between donors) ends and technical variation (variance in replication of culture conditions) begins. In this study, we cultured larval fat bodies from the Japanese rhinoceros beetle, Allomyrina dichotoma, and evaluated, using linear mixed models, the effect of interindividual variation between donors on the reproducibility of the culture. We also performed transcriptome analysis of the hemocyte-like cells mainly seen in the cultures using RNA sequencing and ultrastructural analyses of hemocytes using a transmission electron microscope, revealing that the cultured cells have many characteristics of insect hemocytes.

  4. Primary and Reversible Pisa Syndrome in Juvenile Normal Pressure Hydrocephalus

    PubMed Central

    Leon-Sarmiento, Fidias E.; Pradilla, Gustavo; del Rosario Zambrano, Maria

    2012-01-01

    Objective To report a case of Pisa syndrome in a patient with idiopathic normal pressure hydrocephalus, who had never been exposed to psychotropic medications. Methods A 26 years-old, Colombian, male patient, was referred because he had cognitive abnormalities, gait disturbances and urinary incontinence. This patient also displayed pleurothotonos. Neurofunctional evaluation of sensory and motor integration at peripheral and central nervous system levels were done. Results Pisa syndrome disappeared after spinal tap drainage with further gait, balance and behavioral improvement. A brainstem-thalamocortical deregulation of the central sensory and motor programming, due to the chaotic enlargement of brain ventricles was thought to be the pathophysiological mechanism underlying this case. Conclusion NPH must not be longer considered as an exclusive geriatric disorder. Further, uncommon movement disorders may appear with this disorder, which should be carefully approached to avoid iatrogenic and deleterious pharmacological interventions. PMID:23794788

  5. A method for establishing human primary gastric epithelial cell culture from fresh surgical gastric tissues.

    PubMed

    Aziz, Faisal; Yang, Xuesong; Wen, Qingping; Yan, Qiu

    2015-08-01

    At present, biopsy specimens, cancer cell lines and tissues obtained by gastric surgery are used in the study and analysis of gastric cancer, including the molecular mechanisms and proteomics. However, fibroblasts and other tissue components may interfere with these techniques. Therefore, the present study aimed to develop a procedure for the isolation of viable human gastric epithelial cells from gastric surgical tissues. A method was developed to culture human gastric epithelial cells using fresh, surgically excised tissues and was evaluated using immunocytochemistry, periodic acid-Schiff (PAS) staining and cell viability assays. Low cell growth was observed surrounding the gastric tissue on the seventh day of tissue explant culture. Cell growth subsequently increased, and at 12 days post-explant a high number of pure epithelial cells were detected. The gastric cancer cells exhibited rapid growth with a doubling time of 13-52 h, as compared to normal cells, which had a doubling time of 20-53 h. Immunocytochemical analyses of primary gastric cells revealed positive staining for cytokeratin 18 and 19, which indicated that the culture was comprised of pure epithelial cells and contained no fibroblasts. Furthermore, PAS staining demonstrated that the cultured gastric cells produced neutral mucin. Granulin and carbohydrate antigen 724 staining confirmed the purity of gastric cancer and normal cells in culture. This method of cell culture indicated that the gastric cells in primary culture consisted of mucin-secreting gastric epithelial cells, which may be useful for the study of gastric infection with Helicobacter pylori and gastric cancer.

  6. Efficient establishment of primary fibroblast cultures from the hawksbill sea turtle (Eretmochelys imbricata).

    PubMed

    Fukuda, Tomokazu; Kurita, Jun; Saito, Tomomi; Yuasa, Kei; Kurita, Masanobu; Donai, Kenichiro; Nitto, Hiroshi; Soichi, Makoto; Nishimori, Katsuhiko; Uchida, Takafumi; Isogai, Emiko; Onuma, Manabu; Sone, Hideko; Oseko, Norihisa; Inoue-Murayama, Miho

    2012-12-01

    The hawksbill sea turtle (Eretmochelys imbricata) is a critically endangered species at a risk of extinction. Preservation of the genomic and cellular information of endangered animals is important for future genetic and biological studies. Here, we report the efficient establishment of primary fibroblast cultures from skin tissue of the hawksbill sea turtle. We succeeded in establishing 19 primary cultures from 20 hawksbill sea turtle individuals (a success rate of 95%). These cells exhibited a fibroblast-like morphology and grew optimally at a temperature of 26°C, but experienced a loss of viability when cultured at 37°C. Chromosomal analysis using the primary cells derived here revealed that hawksbill sea turtles have a 2n = 56 karyotype. Furthermore, we showed that our primary cell cultures are free of several fish-related viruses, and this finding is important for preservation purposes. To our knowledge, this report is the first to describe primary cell cultures established from normal tissues of the hawksbill sea turtle. The results will contribute to the preservation of biodiversity, especially for the sea turtles that are critically endangered owing to human activities.

  7. Where Cultural Games Count: The Voices of Primary Classroom Teachers

    ERIC Educational Resources Information Center

    Nabie, Michael Johnson

    2015-01-01

    This study explored Ghanaian primary school teachers' values and challenges of integrating cultural games in teaching mathematics. Using an In-depth conversational interview, ten (10) certificated teachers' voices on the values and challenges of integrating games were examined. Thematic data analysis was applied to the qualitative data from the…

  8. Oncogenic transformation of diverse gastrointestinal tissues in primary organoid culture

    PubMed Central

    Li, Xingnan; Nadauld, Lincoln; Ootani, Akifumi; Corney, David C.; Pai, Reetesh K.; Gevaert, Olivier; Cantrell, Michael A.; Rack, Paul G.; Neal, James T.; Chan, Carol W-M.; Yeung, Trevor; Gong, Xue; Yuan, Jenny; Wilhelmy, Julie; Robine, Sylvie; Attardi, Laura D.; Plevritis, Sylvia K.; Hung, Kenneth E.; Chen, Chang-Zheng; Ji, Hanlee P.; Kuo, Calvin J.

    2014-01-01

    The application of primary organoid cultures containing epithelial and mesenchymal elements to cancer modeling holds promise for combining the accurate multilineage differentiation and physiology of in vivo systems with the facile in vitro manipulation of transformed cell lines. Here, a single air-liquid interface culture method was used without modification to engineer oncogenic mutations into primary epithelial/mesenchymal organoids from mouse colon, stomach and pancreas. Pancreatic and gastric organoids exhibited dysplasia upon KrasG12D expression and/or p53 loss, and readily generated adenocarcinoma upon in vivo transplantation. In contrast, primary colon organoids required combinatorial Apc, p53, KrasG12D and Smad4 mutations for progressive transformation to invasive adenocarcinoma-like histology in vitro and tumorigenicity in vivo, recapitulating multi-hit models of colorectal cancer (CRC), and versus more promiscuous transformation of small intestinal organoids. Colon organoid culture functionally validated the microRNA miR-483 as a dominant driver oncogene at the Insulin-like growth factor-2 (IGF2) 11p15.5 CRC amplicon, inducing dysplasia in vitro and tumorigenicity in vivo. These studies demonstrate the general utility of a highly tractable primary organoid system for cancer modeling and driver oncogene validation in diverse gastrointestinal tissues. PMID:24859528

  9. Alpha particle induced DNA damage and repair in normal cultured thyrocytes of different proliferation status.

    PubMed

    Lyckesvärd, Madeleine Nordén; Delle, Ulla; Kahu, Helena; Lindegren, Sture; Jensen, Holger; Bäck, Tom; Swanpalmer, John; Elmroth, Kecke

    2014-07-01

    Childhood exposure to ionizing radiation increases the risk of developing thyroid cancer later in life and this is suggested to be due to higher proliferation of the young thyroid. The interest of using high-LET alpha particles from Astatine-211 ((211)At), concentrated in the thyroid by the same mechanism as (131)I [1], in cancer treatment has increased during recent years because of its high efficiency in inducing biological damage and beneficial dose distribution when compared to low-LET radiation. Most knowledge of the DNA damage response in thyroid is from studies using low-LET irradiation and much less is known of high-LET irradiation. In this paper we investigated the DNA damage response and biological consequences to photons from Cobolt-60 ((60)Co) and alpha particles from (211)At in normal primary thyrocytes of different cell cycle status. For both radiation qualities the intensity levels of γH2AX decreased during the first 24h in both cycling and stationary cultures and complete repair was seen in all cultures but cycling cells exposed to (211)At. Compared to stationary cells alpha particles were more harmful for cycling cultures, an effect also seen at the pChk2 levels. Increasing ratios of micronuclei per cell nuclei were seen up to 1Gy (211)At. We found that primary thyrocytes were much more sensitive to alpha particle exposure compared with low-LET photons. Calculations of the relative biological effectiveness yielded higher RBE for cycling cells compared with stationary cultures at a modest level of damage, clearly demonstrating that cell cycle status influences the relative effectiveness of alpha particles.

  10. Malignant Transformation of Mouse Primary Keratinocytes by Harvey Sarcoma Virus and Its Modulation by Surrounding Normal Cells

    NASA Astrophysics Data System (ADS)

    Dotto, Gian Paolo; Weinberg, Robert A.; Ariza, Aurelio

    1988-09-01

    The activated ras oncogene that is present in Harvey sarcoma virus is able to induce malignant transformation of pure cultures of mouse primary keratinocytes. Malignant transformation of these cells is demonstrated by their ability to form carcinomas when grafted back onto syngeneic animals. However, expression of the malignant phenotype by the ras-transformed keratinocytes is drastically inhibited by the presence of normal dermal fibroblasts. This inhibitory effect depends on the ratio of fibroblasts to keratinocytes. It can be observed with mitomycin C-treated growth-arrested dermal fibroblasts and not with other cells, such as normal keratinocytes or established fibroblasts. Thus, a cellular environment approximating normal tissue can suppress tumor formation triggered by a single oncogene.

  11. Microbiologic and clinical value of primary broth cultures of wound specimens collected with swabs.

    PubMed Central

    Silletti, R P; Ailey, E; Sun, S; Tang, D

    1997-01-01

    In order to assess the microbiologic and clinical value of primary broth culture of wound specimens collected with swabs and submitted to the laboratory in transport medium, we compared the results of primary agar culture with the results of a corresponding primary broth culture for 344 aerobic specimens and 176 anaerobic specimens. While 8.7% (45 of 520) of the specimens yielded organisms from the primary broth culture that were not recovered from the corresponding primary agar culture, only 5.0% (26 of 520) of the specimens yielded organisms from the primary broth culture other than Staphylococcus epidermidis, viridans group streptococci, and Corynebacterium spp. Moreover, the primary broth culture of only 0.6% (3 of 520) of the specimens yielded organisms not recovered from the primary agar culture that caused a change in the therapy of the patient. Our conclusion is that primary broth cultures are unnecessary for the processing of wound specimens properly collected with swabs. PMID:9230370

  12. Cross-cultural aspects of depression management in primary care.

    PubMed

    Hails, Katherine; Brill, Charlotte D; Chang, Trina; Yeung, Albert; Fava, Maurizio; Trinh, Nhi-Ha

    2012-08-01

    Major depressive disorder (MDD) is a prevalent illness in minority populations. Minority patients with MDD are often unrecognized and untreated. This review examines promising interventions to address MDD in primary care settings, where minority groups are more likely to seek care. Since 2010, eleven interventions have been developed to address patient-specific and provider-specific barriers, many of which are adaptations of the collaborative care model. Other promising interventions include cultural tailoring of the collaborative care model, as well as the addition of telepsychiatry, motivational interviewing, cultural consultation, and innovations in interpreting. Overall, collaborative care was found feasible and improved satisfaction and treatment engagement of depressed minority patients in primary care. It remains inconclusive whether these newer intervention models improve MDD treatment outcomes. Future research will be needed to establish the effectiveness of these intervention models in improving the treatment outcomes of minority populations with MDD.

  13. Primary Postnatal Dorsal Root Ganglion Culture from Conventionally Slaughtered Calves

    PubMed Central

    Fadda, A.; Bärtschi, M.; Hemphill, A.; Widmer, H. R.; Zurbriggen, A.; Perona, P.; Vidondo, B.; Oevermann, A.

    2016-01-01

    Neurological disorders in ruminants have an important impact on veterinary health, but very few host-specific in vitro models have been established to study diseases affecting the nervous system. Here we describe a primary neuronal dorsal root ganglia (DRG) culture derived from calves after being conventionally slaughtered for food consumption. The study focuses on the in vitro characterization of bovine DRG cell populations by immunofluorescence analysis. The effects of various growth factors on neuron viability, neurite outgrowth and arborisation were evaluated by morphological analysis. Bovine DRG neurons are able to survive for more than 4 weeks in culture. GF supplementation is not required for neuronal survival and neurite outgrowth. However, exogenously added growth factors promote neurite outgrowth. DRG cultures from regularly slaughtered calves represent a promising and sustainable host specific model for the investigation of pain and neurological diseases in bovines. PMID:27936156

  14. Surface markers on human lymphocytes: studies of normal subjects and of patients with primary immunodeficiencies

    PubMed Central

    Aiuti, F.; Lacava, V.; Garofalo, J. A.; D'Amelio, R.; D'Asero, C.

    1973-01-01

    Peripheral blood lymphocytes of twenty normal controls and twelve patients with primary immunodeficiencies were examined for surface membrane Ig and receptors for C3 complement (B cell markers) and for spontaneous rosette formation with sheep erythrocytes (T cell markers). In patients with defects in T cell function no lymphocytes forming spontaneous rosettes were seen. In patients with B cell deficiency they were normal or increased. Lymphocytes with membrane immunoglobulins were normal in patients with T cell defect and absent in patients with severe agammaglobulinaemia. Lymphocytes with receptors for C3 complement were increased in patients with T defect and normal in patients with most other forms of immunodeficiency studied. PMID:4587827

  15. Organizational culture in the primary healthcare setting of Cyprus

    PubMed Central

    2013-01-01

    Background The concept of organizational culture is important in understanding the behaviour of individuals in organizations as they manage external demands and internal social changes. Cyprus healthcare system is under restructuring and soon a new healthcare scheme will be implemented starting at the Primary Healthcare (PHC) level. The aim of the study was to investigate the underlying culture encountered in the PHC setting of Cyprus and to identify possible differences in desired and prevailing cultures among healthcare professionals. Methods The population of the study included all general practitioners (GPs) and nursing staff working at the 42 PHC centres throughout the island. The shortened version of the Organizational Culture Profile questionnaire comprising 28 statements on organizational values was used in the study. The instrument was already translated and validated in Greek and cross-cultural adaptation was performed. Participants were required to indicate the organization’s characteristic cultural values orientation along a five-point Likert scale ranging from “Very Much = 1” to “Not at all= 5”. Statistical analysis was performed using SPSS 16.0. Student t-test was used to compare means between two groups of variables whereas for more than two groups analysis of variance (ANOVA) was applied. Results From the total of 306 healthcare professionals, 223 participated in the study (72.9%). The majority of participants were women (75.3%) and mean age was 42.6 ± 10.7 years. Culture dimension “performance orientation” was the desired culture among healthcare professionals (mean: 1.39 ± 0.45). “Supportiveness” and “social responsibility” were the main cultures encountered in PHC (means: 2.37 ± 0.80, 2.38 ± 0.83). Statistical significant differences were identified between desired and prevailing cultures for all culture dimensions (p= 0.000). Conclusions This was the first study performed in Cyprus assessing organizational culture in

  16. Rhamnolipids elicit the same cytotoxic sensitivity between cancer cell and normal cell by reducing surface tension of culture medium.

    PubMed

    Jiang, Lifang; Shen, Chong; Long, Xuwei; Zhang, Guoliang; Meng, Qin

    2014-12-01

    Biosurfactant rhamnolipids have been claimed to show biological activities of inhibiting the proliferation of cancer cells. In this study, the cytotoxicity of rhamnolipids was examined on four cancer cells (HepG2, Caco-2, Hela, MCF-7 cells) and two normal cells (HK-2 cell, primary hepatocyte). Interestingly, both cancer cells and normal cells exhibited similar sensitivities to the addition of rhamnolipids in culture medium, and the cytotoxicity was largely attenuated by the presence of fetal bovine serum (FBS) in culture medium. In correlation of the mono-/di-rhamnolipid cytotoxicity with the surface tension of culture medium, it was found that rhamnolipids triggered cytotoxicity whenever the surface tension of culture medium decreased below 41 mN/m irrespective of the FBS content in culture medium, cell line, or rhamnolipid congener. Similarly, each chemical surfactant (Tween-80, sodium dodecyl sulfate, and sodium dodecyl benzene sulfonate) could cause cytotoxicity on HepG2 cells whenever its addition made the surface tension under 41 mN/m in culture medium with or without the presence of FBS. It seems that rhamnolipids, like chemical surfactants, exhibited cytotoxicity by reducing the surface tension of culture medium rather than by changing its specific molecular structure, which had no selection on tumor cells. This study could offer helps to correct the misleading biological activity of rhamnolipids and to avoid the possible large wastes of time and expenses on developing the applications in antitumor drugs.

  17. Effects of Ranolazine on Astrocytes and Neurons in Primary Culture.

    PubMed

    Aldasoro, Martin; Guerra-Ojeda, Sol; Aguirre-Rueda, Diana; Mauricio, M Dolores; Vila, Jose M; Marchio, Patricia; Iradi, Antonio; Aldasoro, Constanza; Jorda, Adrian; Obrador, Elena; Valles, Soraya L

    2016-01-01

    Ranolazine (Rn) is an antianginal agent used for the treatment of chronic angina pectoris when angina is not adequately controlled by other drugs. Rn also acts in the central nervous system and it has been proposed for the treatment of pain and epileptic disorders. Under the hypothesis that ranolazine could act as a neuroprotective drug, we studied its effects on astrocytes and neurons in primary culture. We incubated rat astrocytes and neurons in primary cultures for 24 hours with Rn (10-7, 10-6 and 10-5 M). Cell viability and proliferation were measured using trypan blue exclusion assay, MTT conversion assay and LDH release assay. Apoptosis was determined by Caspase 3 activity assay. The effects of Rn on pro-inflammatory mediators IL-β and TNF-α was determined by ELISA technique, and protein expression levels of Smac/Diablo, PPAR-γ, Mn-SOD and Cu/Zn-SOD by western blot technique. In cultured astrocytes, Rn significantly increased cell viability and proliferation at any concentration tested, and decreased LDH leakage, Smac/Diablo expression and Caspase 3 activity indicating less cell death. Rn also increased anti-inflammatory PPAR-γ protein expression and reduced pro-inflammatory proteins IL-1 β and TNFα levels. Furthermore, antioxidant proteins Cu/Zn-SOD and Mn-SOD significantly increased after Rn addition in cultured astrocytes. Conversely, Rn did not exert any effect on cultured neurons. In conclusion, Rn could act as a neuroprotective drug in the central nervous system by promoting astrocyte viability, preventing necrosis and apoptosis, inhibiting inflammatory phenomena and inducing anti-inflammatory and antioxidant agents.

  18. Effects of Ranolazine on Astrocytes and Neurons in Primary Culture

    PubMed Central

    Aldasoro, Martin; Guerra-Ojeda, Sol; Aguirre-Rueda, Diana; Mauricio, Mª Dolores; Vila, Jose Mª; Marchio, Patricia; Iradi, Antonio; Aldasoro, Constanza; Jorda, Adrian; Obrador, Elena; Valles, Soraya L.

    2016-01-01

    Ranolazine (Rn) is an antianginal agent used for the treatment of chronic angina pectoris when angina is not adequately controlled by other drugs. Rn also acts in the central nervous system and it has been proposed for the treatment of pain and epileptic disorders. Under the hypothesis that ranolazine could act as a neuroprotective drug, we studied its effects on astrocytes and neurons in primary culture. We incubated rat astrocytes and neurons in primary cultures for 24 hours with Rn (10−7, 10−6 and 10−5 M). Cell viability and proliferation were measured using trypan blue exclusion assay, MTT conversion assay and LDH release assay. Apoptosis was determined by Caspase 3 activity assay. The effects of Rn on pro-inflammatory mediators IL-β and TNF-α was determined by ELISA technique, and protein expression levels of Smac/Diablo, PPAR-γ, Mn-SOD and Cu/Zn-SOD by western blot technique. In cultured astrocytes, Rn significantly increased cell viability and proliferation at any concentration tested, and decreased LDH leakage, Smac/Diablo expression and Caspase 3 activity indicating less cell death. Rn also increased anti-inflammatory PPAR-γ protein expression and reduced pro-inflammatory proteins IL-1 β and TNFα levels. Furthermore, antioxidant proteins Cu/Zn-SOD and Mn-SOD significantly increased after Rn addition in cultured astrocytes. Conversely, Rn did not exert any effect on cultured neurons. In conclusion, Rn could act as a neuroprotective drug in the central nervous system by promoting astrocyte viability, preventing necrosis and apoptosis, inhibiting inflammatory phenomena and inducing anti-inflammatory and antioxidant agents. PMID:26950436

  19. Culturing primary rat inner medullary collecting duct cells.

    PubMed

    Faust, Dörte; Geelhaar, Andrea; Eisermann, Beate; Eichhorst, Jenny; Wiesner, Burkhard; Rosenthal, Walter; Klussmann, Enno; Klussman, Enno

    2013-06-21

    Arginine-vasopressin (AVP) facilitates water reabsorption by renal collecting duct principal cells and thereby fine-tunes body water homeostasis. AVP binds to vasopressin V2 receptors (V2R) on the surface of the cells and thereby induces synthesis of cAMP. This stimulates cellular signaling processes leading to changes in the phosphorylation of the water channel aquaporin-2 (AQP2). Protein kinase A phoshorylates AQP2 and thereby triggers the translocation of AQP2 from intracellular vesicles into the plasma membrane facilitating water reabsorption from primary urine. Aberrations of AVP release from the pituitary or AVP-activated signaling in principal cells can cause central or nephrogenic diabetes insipidus, respectively; an elevated blood plasma AVP level is associated with cardiovascular diseases such as chronic heart failure and the syndrome of inappropriate antidiuretic hormone secretion. Here, we present a protocol for cultivation of primary rat inner medullary collecting duct (IMCD) cells, which express V2R and AQP2 endogenously. The cells are suitable for elucidating molecular mechanisms underlying the control of AQP2 and thus to discover novel drug targets for the treatment of diseases associated with dysregulation of AVP-mediated water reabsorption. IMCD cells are obtained from rat renal inner medullae and are used for experiments six to eight days after seeding. IMCD cells can be cultured in regular cell culture dishes, flasks and micro-titer plates of different formats, the procedure only requires a few hours, and is appropriate for standard cell culture laboratories.

  20. Culturing Primary Rat Inner Medullary Collecting Duct Cells

    PubMed Central

    Faust, Dörte; Geelhaar, Andrea; Eisermann, Beate; Eichhorst, Jenny; Wiesner, Burkhard; Rosenthal, Walter; Klussman, Enno

    2013-01-01

    Arginine-vasopressin (AVP) facilitates water reabsorption by renal collecting duct principal cells and thereby fine-tunes body water homeostasis. AVP binds to vasopressin V2 receptors (V2R) on the surface of the cells and thereby induces synthesis of cAMP. This stimulates cellular signaling processes leading to changes in the phosphorylation of the water channel aquaporin-2 (AQP2). Protein kinase A phoshorylates AQP2 and thereby triggers the translocation of AQP2 from intracellular vesicles into the plasma membrane facilitating water reabsorption from primary urine. Aberrations of AVP release from the pituitary or AVP-activated signaling in principal cells can cause central or nephrogenic diabetes insipidus, respectively; an elevated blood plasma AVP level is associated with cardiovascular diseases such as chronic heart failure and the syndrome of inappropriate antidiuretic hormone secretion. Here, we present a protocol for cultivation of primary rat inner medullary collecting duct (IMCD) cells, which express V2R and AQP2 endogenously. The cells are suitable for elucidating molecular mechanisms underlying the control of AQP2 and thus to discover novel drug targets for the treatment of diseases associated with dysregulation of AVP-mediated water reabsorption. IMCD cells are obtained from rat renal inner medullae and are used for experiments six to eight days after seeding. IMCD cells can be cultured in regular cell culture dishes, flasks and micro-titer plates of different formats, the procedure only requires a few hours, and is appropriate for standard cell culture laboratories. PMID:23852264

  1. Effect of primary culture medium type for culture of canine fibroblasts on production of cloned dogs.

    PubMed

    Kim, Geon A; Oh, Hyun Ju; Kim, Min Jung; Jo, Young Kwang; Choi, Jin; Kim, Jin Wook; Lee, Tae Hee; Lee, Byeong Chun

    2015-09-01

    Fibroblasts are common source of donor cells for SCNT. It is suggested that donor cells' microenvironment, including the primary culture, affects development of reconstructed embryos. To prove this, canine embryos were cloned with fibroblasts that were cultured in two different primary media (RCMEp vs. Dulbecco's modified Eagle's medium [DMEM]) and in vivo developments were compared with relative amount of stemness, reprogramming, apoptosis gene transcripts, and telomerase activity. Donor cells cultured in RCMEp contained a significantly higher amount of SOX2, NANOG, DPPA2, REXO1, HDAC, DNMT1, MECP2 and telomerase activity than those cultured in DMEM (P < 0.05). In vivo developmental potential of cloned embryos with donor cells cultured in RCMEp had a higher birth rate than that of embryos derived from DMEM (P < 0.05). The culture medium can induce changes in gene expression of donor cells and telomerase activity, and these alterations can also affect in vivo developmental competence of the cloned embryos.

  2. Antisense oligodeoxynucleotide to the cystic fibrosis gene inhibits anion transport in normal cultured sweat duct cells

    SciTech Connect

    Sorscher, E.J.; Kirk, K.L.; Weaver, M.L.; Jilling, T.; Blalock, J.E.; LeBoeuf, R.D. )

    1991-09-01

    The authors have tested the hypothesis that the cystic fibrosis (CF) gene product, called the CF transmembrane conductance regulator (CFTR), mediates anion transport in normal human sweat duct cells. Sweat duct cells in primary culture were treated with oligodeoxynucleotides that were antisense to the CFTR gene transcript in order to block the expression of the wild-type CFTR. Anion transport in CFTR transcript antisense-treated cells was then assessed with a halide-specific dye, 6-methoxy-N-(3-sulfopropryl)quinolinium, and fluorescent digital imaging microscopy to monitor halide influx and efflux from single sweat duct cells. Antisense oligodeoxynucleotide treatment for 24 hr virtually abolished Cl{sup {minus}} transport in sweat duct cells compared with untreated cells or control cells treated with sense oligodeoxynucleotides. Br{sup {minus}} uptake into sweat duct cells was also blocked after a 24-hr CFTR transcript antisense treatments, but not after treatments for only 4 hr. Lower concentrations of antisense oligodeoxynucleotides were less effective at inhibiting Cl{sup {minus}} transport. These results indicate that oligodeoxynucleotides that are antisense to CFTR transcript inhibit sweat duct Cl{sup {minus}} permeability in both a time-dependent and dose-dependent manner. This approach provides evidence that inhibition of the expression of the wild-type CFTR gene in a normal, untransfected epithelial cell results in an inhibition of Cl{sup {minus}} permeability.

  3. Primary cell cultures of bovine colon epithelium: isolation and cell culture of colonocytes.

    PubMed

    Föllmann, W; Weber, S; Birkner, S

    2000-10-01

    Epithelial cells from bovine colon were isolated by mechanical preparation combined with an enzymatic digestion from colon specimens derived from freshly slaughtered animals. After digestion with collagenase I, the isolated tissue was centrifuged on a 2% D-sorbitol gradient to separate epithelial crypts which were seeded in collagen I-coated culture flasks. By using colon crypts and omitting the seeding of single cells a contamination by fibroblasts was prevented. The cells proliferated under the chosen culture conditions and formed monolayer cultures which were maintained for several weeks, including subcultivation steps. A population doubling time of about 21 hr was estimated in the log phase of the corresponding growth curve. During the culture period the cells were characterized morphologically and enzymatically. By using antibodies against cytokeratine 7 and 13 the isolated cells were identified as cells of epithelial origin. Antibodies against vimentin served as negative control. Morphological features such as microvilli, desmosomes and tight junctions, which demonstrated the ability of the cultured cells to restore an epithelial like monolayer, were shown by ultrastructural investigations. The preservation of the secretory function of the cultured cells was demonstrated by mucine cytochemistry with alcian blue staining. A stable expression of enzyme activities over a period of 6 days in culture occurred for gamma-glutamyltranspeptidase, acid phosphatase and NADH-dehydrogenase activity under the chosen culture conditions. Activity of alkaline phosphatase decreased to about 50% of basal value after 6 days in culture. Preliminary estimations of the metabolic competence of these cells revealed cytochrome P450 1A1-associated EROD activity in freshly isolated cells which was stable over 5 days in cultured cells. Then activity decreased completely. This culture system with primary epithelial cells from the colon will be used further as a model for the colon

  4. A reporter assay for target validation in primary neuronal cultures.

    PubMed

    Pollio, G; Roncarati, R; Seredenina, T; Terstappen, G C; Caricasole, A

    2008-07-15

    The deposition of beta-amyloid peptides (Abeta42 and Abeta40) in neuritic plaques is one of the hallmarks of Alzheimer's disease (AD), and genes modulating their brain levels and neuronal effects could result in future disease modifying therapies. The causal association of candidate targets with AD is of paramount importance in current drug discovery, as a lack of efficacy of many candidate drugs is often due to inadequate validation of their pharmacological target. In Alzheimer's as well as in other neurodegenerative diseases, in vitro target validation is hampered by the difficulty of transfecting primary neuronal cultures and assaying the effects of genes on neuronal viability. Here we describe a rapid, sensitive and simple reporter-based assay for the validation of genes putatively associated with Abeta-mediated neurotoxicity, which can in principle be extended to the validation of targets in the context of other neuronal insults. The assay is suitable for the generation of robust and reproducible data in primary neuronal cultures allowing the dissection at a molecular level of complex pathways activated by the toxic insult in a cellular context that more closely represents the real disease situation.

  5. Zinc Modulates Nanosilver-Induced Toxicity in Primary Neuronal Cultures.

    PubMed

    Ziemińska, Elżbieta; Strużyńska, Lidia

    2016-02-01

    Silver nanoparticles (NAg) have recently become one of the most commonly used nanomaterials. Since the ability of nanosilver to enter the brain has been confirmed, there has been a need to investigate mechanisms of its neurotoxicity. We previously showed that primary neuronal cultures treated with nanosilver undergo destabilization of calcium homeostasis via a mechanism involving glutamatergic NMDA receptors. Considering the fact that zinc interacts with these receptors, the aim of the present study was to examine the role of zinc in mechanisms of neuronal cell death in primary cultures. In cells treated with nanosilver, we noted an imbalance between extracellular and intracellular zinc levels. Thus, the influence of zinc deficiency and supplementation on nanosilver-evoked cytotoxicity was investigated by treatment with TPEN (a chelator of zinc ions), or ZnCl(2), respectively. Elimination of zinc leads to complete death of nanosilver-treated CGCs. In contrast, supplementation with ZnCl(2) increases viability of CGCs in a dose-dependent manner. Addition of zinc provided protection against the extra/intracellular calcium imbalance in a manner similar to MK-801, an antagonist of NMDA receptors. Zinc chelation by TPEN decreases the mitochondrial potential and dramatically increases the rate of production of reactive oxygen species. Our results indicate that zinc supplementation positively influences nanosilver-evoked changes in CGCs. This is presumed to be due to an inhibitory effect on NMDA-sensitive calcium channels.

  6. Toxicity of redox cycling pesticides in primary mesencephalic cultures.

    PubMed

    Bonneh-Barkay, Dafna; Langston, William J; Di Monte, Donato A

    2005-01-01

    A loss of nigrostriatal dopaminergic neurons is the primary neurodegenerative feature of Parkinson's disease. Paraquat, a known redox cycling herbicide, has recently been shown to kill selectively nigrostriatal dopaminergic cells in the mouse model. The purpose of this study was to test the ability of paraquat and other redox cycling pesticides to damage dopaminergic neurons in primary mesencephalic cultures. Addition of paraquat, diquat, or benzyl viologen to mesencephalic cultures induced morphological changes (e.g., dystrophic neuronal processes) consistent with dopaminergic cell injury. The three pesticides also caused cell death as assessed by a reduction of the number of tyrosine hydroxylase-immunoreactive neurons and a dose-dependent decrease in [(3)H]dopamine uptake. Quite interestingly, diquat and benzyl viologen were significantly more toxic than paraquat, probably reflecting their more pronounced ability to trigger redox cycling reactions. The data support a role of redox cycling as a mechanism of dopaminergic cell degeneration and suggest that the property of redox cycling should be taken into consideration when evaluating putative environmental risk factors for Parkinson's disease.

  7. Early loss of mitochondrial inner transmembrane potential in khat-induced cell death of primary normal human oral cells.

    PubMed

    Lukandu, Ochiba M; Bredholt, Therese; Neppelberg, Evelyn; Gjertsen, Bjørn T; Johannessen, Anne C; Vintermyr, Olav K; Costea, Daniela Elena

    2009-09-19

    Previous studies suggest the use of khat, a psychostimulant plant used by millions of people in Middle East and Africa, as risk factor for oral cancer. We previously reported that khat is able to induce adverse affects, as cell cycle arrest and apoptosis, in normal human oral cells cultured in vitro. This study further investigates the more specific role played by mitochondria in khat-induced cell death and the kinetics of the events involved in this process. Exposure of primary normal human oral keratinocytes and fibroblasts to khat extract resulted in a swift and sustained decrease of the mitochondrial inner transmembrane potential occurring within 0.5-1h. Loss of mitochondrial membrane potential preceded all other biochemical and morphologic changes, and was associated with a significant decrease in cell survival. Subsequently, apoptosis-inducing factor was released from mitochondria into cytosol and relocated to nucleus. Cyclosporine A and bongkrekic acid delayed both the loss of mitochondrial inner transmembrane potential and the onset of cell death. This study describes a novel mechanism of khat-induced cell death in primary normal oral keratinocytes and fibroblasts involving an early pivotal effect on mitochondrial function and integrity.

  8. Accumulation of silver nanoparticles by cultured primary brain astrocytes

    NASA Astrophysics Data System (ADS)

    Luther, Eva M.; Koehler, Yvonne; Diendorf, Joerg; Epple, Matthias; Dringen, Ralf

    2011-09-01

    Silver nanoparticles (AgNP) are components of various food industry products and are frequently used for medical equipment and materials. Although such particles enter the vertebrate brain, little is known on their biocompatibility for brain cells. To study the consequences of an AgNP exposure of brain cells we have treated astrocyte-rich primary cultures with polyvinylpyrrolidone (PVP)-coated AgNP. The incubation of cultured astrocytes with micromolar concentrations of AgNP for up to 24 h resulted in a time- and concentration-dependent accumulation of silver, but did not compromise the cell viability nor lower the cellular glutathione content. In contrast, the incubation of astrocytes for 4 h with identical amounts of silver as AgNO3 already severely compromised the cell viability and completely deprived the cells of glutathione. The accumulation of AgNP by astrocytes was proportional to the concentration of AgNP applied and significantly lowered by about 30% in the presence of the endocytosis inhibitors chloroquine or amiloride. Incubation at 4 °C reduced the accumulation of AgNP by 80% compared to the values obtained for cells that had been exposed to AgNP at 37 °C. These data demonstrate that viable cultured brain astrocytes efficiently accumulate PVP-coated AgNP in a temperature-dependent process that most likely involves endocytotic pathways.

  9. Accumulation of silver nanoparticles by cultured primary brain astrocytes.

    PubMed

    Luther, Eva M; Koehler, Yvonne; Diendorf, Joerg; Epple, Matthias; Dringen, Ralf

    2011-09-16

    Silver nanoparticles (AgNP) are components of various food industry products and are frequently used for medical equipment and materials. Although such particles enter the vertebrate brain, little is known on their biocompatibility for brain cells. To study the consequences of an AgNP exposure of brain cells we have treated astrocyte-rich primary cultures with polyvinylpyrrolidone (PVP)-coated AgNP. The incubation of cultured astrocytes with micromolar concentrations of AgNP for up to 24 h resulted in a time- and concentration-dependent accumulation of silver, but did not compromise the cell viability nor lower the cellular glutathione content. In contrast, the incubation of astrocytes for 4 h with identical amounts of silver as AgNO(3) already severely compromised the cell viability and completely deprived the cells of glutathione. The accumulation of AgNP by astrocytes was proportional to the concentration of AgNP applied and significantly lowered by about 30% in the presence of the endocytosis inhibitors chloroquine or amiloride. Incubation at 4 °C reduced the accumulation of AgNP by 80% compared to the values obtained for cells that had been exposed to AgNP at 37 °C. These data demonstrate that viable cultured brain astrocytes efficiently accumulate PVP-coated AgNP in a temperature-dependent process that most likely involves endocytotic pathways.

  10. Expression, localisation and functional activation of NFAT-2 in normal human skin, psoriasis, and cultured keratocytes

    PubMed Central

    Al-Daraji, Wael I; Malak, Tamer T.; Prescott, Richard J.; Abdellaoui, Adel; Ali, Mahmud M.; Dabash, Tarek; Zelger, Bettina G.; Zelger, Bernhard

    2009-01-01

    Ciclosporin A (CsA) is widely utilized for the treatment of inflammatory skin diseases such as psoriasis. The therapeutic effects of CsA are thought to be mediated via its immunosuppressive action on infiltrating lymphocytes in skin lesions. CsA and tacrolimus block T cell activation by inhibiting the phosphatase calcineurin and preventing translocation from the cytoplasm to the nucleus of the transcription factor Nuclear Factor of Activated T cells (NFAT). As calcineurin and NFAT 1 have been shown to be functionally active in cultured human keratocytes, expression of other NFAT family members such as NFAT-2 and possible functional activation was investigated in human keratocytes. RT-PCR and Western Analysis were used to investigate the presence of NFAT-2 mRNA and protein in human keratocytes. Tissue culture of human keratocytes and immunostaining of cells on coverslips and confocal microscopy were used to assess the degree of nuclear localisation of NFAT-2 in cultured cells. Keratome biopsies were taken from patients with psoriasis (lesional and non-lesional skin) and normal skin and immunohistochemistry was used to assess the NFAT-2 localisation in these biopsies using a well characterized anti-NFAT-2 antibody. The NFAT-2 mRNA and protein expression was demonstrated using RT-PCR and Western blotting. Moreover, the expression of NFAT-2 in normal skin, non-lesional and lesional psoriasis showed a striking basal staining suggesting a role for NFAT-2 in keratocytes proliferation. A range of cell types in the skin express NFAT-2. The expression of NFAT-2 in human keratocytes and response to different agonists provides perhaps a unique opportunity to examine the regulation, subcellular localization and kinetics of translocation of different NFATs in primary cultured human cells. In these experiments the author assessed the expression, localization of NFAT-2 in cultured human keratocytes and measured the degree of nuclear localisaion of NFAT-2 using immunofluorescence

  11. Assessment of cell viability in primary neuronal cultures.

    PubMed

    Aras, Mandar A; Hartnett, Karen A; Aizenman, Elias

    2008-07-01

    This unit contains five protocols for assaying cell viability in vitro using primary neuronal cultures, including a novel method for use with transfected neurons. Three of the assays are based on the principle that cell death cascades alter membrane permeability. The lactate dehydrogenase (LDH) release assay measures the amount of the cytoplasmic enzyme released into the bathing medium, while the trypan blue and propidium iodide assays measure the ability of cells to exclude dye from their cytoplasm. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay measures the mitochondrial activity of viable cells by quantifying the conversion of the tetrazolium salt to its formazan product. Finally, the fifth assay details the measurement of luciferase expression as an indication of neuronal viability within a relatively small population of transfected neurons.

  12. ATP stimulates calcium influx in primary astrocyte cultures

    SciTech Connect

    Neary, J.T.; van Breemen, C.; Forster, E.; Norenberg, L.O.; Norenberg, M.D.

    1988-12-30

    The effect of ATP and other purines on /sup 45/Ca uptake was studied in primary cultures of rat astrocytes. Treatment of the cells with ATP for 1 to 30 min brought about an increase in cellular /sup 45/Ca. Stimulation of calcium influx by ATP was investigated using a 90 sec exposure to /sup 45/Ca and over a concentration range of 0.1 nM to 3 mM; a biphasic dose-response curve was obtained with EC50 values of 0.3 nM and 9 uM, indicating the presence of low and high affinity purinergic binding sites. Similar levels of /sup 45/Ca influx at 90 sec were observed with ATP, ADP and adenosine (all at 100 uM). Prior treatment of the cultures with LaCl3 blocked the purine-induced /sup 45/Ca influx. These findings indicate that one pathway for calcium entry in astrocytes involves purinergic receptor-operated, calcium channels.

  13. Regulation of human renin expression in chorion cell primary cultures

    SciTech Connect

    Duncan, K.G.; Haidar, M.A.; Baxter, J.D.; Reudelhuber, T.L. )

    1990-10-01

    The human renin gene is expressed in the kidney, placenta, and several other sites. The release of renin or its precursor, prorenin, can be affected by several regulatory agents. In this study, primary cultures of human placental cells were used to examine the regulation of prorenin release and renin mRNA levels and of the transfected human renin promoter linked to chloramphenicol acetyltransferase reporter sequences. Treatment of the cultures with a calcium ionophore alone, calcium ionophore plus forskolin (that activates adenylate cyclase), or forskolin plus a phorbol ester increased prorenin release and renin mRNA levels 1.3{endash} to 6{endash}fold, but several classes of steroids did not affect prorenin secretion or renin RNA levels. These results suggest that (i) the first 584 base pairs of the renin gene 5'{endash}flanking DNA do not contain functional glucocorticoid or estrogen response elements, (ii) placental prorenin release and renin mRNA are regulated by calcium ion and by the combinations of cAMP with either C kinase or calcium ion, and (iii) the first 100 base pairs of the human renin 5'{endash}flanking DNA direct accurate initiation of transcription and can be regulated by cAMP. Thus, some control of renin release in the placenta (and by inference in other tissues) occurs via transcriptional influences on its promoter.

  14. Political, cultural and economic foundations of primary care in Europe.

    PubMed

    Kringos, Dionne S; Boerma, Wienke G W; van der Zee, Jouke; Groenewegen, Peter P

    2013-12-01

    This article explores various contributing factors to explain differences in the strength of the primary care (PC) structure and services delivery across Europe. Data on the strength of primary care in 31 European countries in 2009/10 were used. The results showed that the national political agenda, economy, prevailing values, and type of healthcare system are all important factors that influence the development of strong PC. Wealthier countries are associated with a weaker PC structure and lower PC accessibility, while Eastern European countries seemed to have used their growth in national income to strengthen the accessibility and continuity of PC. Countries governed by left-wing governments are associated with a stronger PC structure, accessibility and coordination of PC. Countries with a social-security based system are associated with a lower accessibility and continuity of PC; the opposite is true for transitional systems. Cultural values seemed to affect all aspects of PC. It can be concluded that strengthening PC means mobilising multiple leverage points, policy options, and political will in line with prevailing values in a country.

  15. Elemental composition of normal primary tooth enamel analyzed with XRMA and SIMS.

    PubMed

    Sabel, Nina; Dietz, Wolfram; Lundgren, Ted; Nietzsche, Sandor; Odelius, Hans; Rythén, Marianne; Rizell, Sara; Robertson, Agneta; Norén, Jörgen G; Klingberg, Gunilla

    2009-01-01

    There is an interest to analyze the chemical composition of enamel in teeth from patients with different developmental disorders or syndromes and evaluate possible differences compared to normal composition. For this purpose, it is essential to have reference material. The aim of this study was to, by means of X-ray micro analyses (XRMA) and secondary ion mass spectrometry (SIMS), present concentration gradients for C, O, P and Ca and F, Na, Mg, Cl, K and Sr in normal enamel of primary teeth from healthy individuals. 36 exfoliated primary teeth from 36 healthy children were collected, sectioned, and analyzed in the enamel and dentin with X-ray micro analyses for the content of C, O, P and Ca and F, Na MgCl, K and Sr. This study has supplied reference data for C, O, P and Ca in enamel in primary teeth from healthy subjects. No statistically significant differences in the elemental composition were found between incisors and molars.The ratio Ca/P is in concordance with other studies. Some elements have shown statistically significant differences between different levels of measurement. These results may be used as reference values for research on the chemical composition of enamel and dentin in primary teeth from patients with different conditions and/or syndromes.

  16. Long-term culture and functional characterization of follicular cells from adult normal human thyroids.

    PubMed Central

    Curcio, F; Ambesi-Impiombato, F S; Perrella, G; Coon, H G

    1994-01-01

    We have obtained long-term cultures of differentiated proliferating follicular cells from normal adult human thyroid glands. In vitro growth of such human cells has been sustained by a modified F-12 medium, supplemented with bovine hypothalamus and pituitary extracts and no added thyrotropin. Cultures have been expanded, cloned, frozen, successfully retrieved, and characterized. Functional characterization of these cells shows constitutive thyroglobulin production and release and thyrotropin-dependent adenosine 3',5'-cyclic monophosphate production, the latter apparently not associated with significant increases in DNA synthesis or cell proliferation. Genetic characterization of these cells by chromosome counting showed the normal diploid chromosome number. The ability to cultivate differentiated human thyroid follicular cells in long-term culture opens possibilities for investigating the transduction pathways of thyrotropin stimulation in normal and pathological human tissues, developing clinically relevant in vitro assays, and considering cellular and molecular therapies. Images PMID:8090760

  17. Interspecies differences in metabolism of arsenic by cultured primary hepatocytes

    SciTech Connect

    Drobna, Zuzana; Walton, Felecia S.; Harmon, Anne W.; Thomas, David J.; Styblo, Miroslav

    2010-05-15

    Biomethylation is the major pathway for the metabolism of inorganic arsenic (iAs) in many mammalian species, including the human. However, significant interspecies differences have been reported in the rate of in vivo metabolism of iAs and in yields of iAs metabolites found in urine. Liver is considered the primary site for the methylation of iAs and arsenic (+3 oxidation state) methyltransferase (As3mt) is the key enzyme in this pathway. Thus, the As3mt-catalyzed methylation of iAs in the liver determines in part the rate and the pattern of iAs metabolism in various species. We examined kinetics and concentration-response patterns for iAs methylation by cultured primary hepatocytes derived from human, rat, mice, dog, rabbit, and rhesus monkey. Hepatocytes were exposed to [{sup 73}As]arsenite (iAs{sup III}; 0.3, 0.9, 3.0, 9.0 or 30 nmol As/mg protein) for 24 h and radiolabeled metabolites were analyzed in cells and culture media. Hepatocytes from all six species methylated iAs{sup III} to methylarsenic (MAs) and dimethylarsenic (DMAs). Notably, dog, rat and monkey hepatocytes were considerably more efficient methylators of iAs{sup III} than mouse, rabbit or human hepatocytes. The low efficiency of mouse, rabbit and human hepatocytes to methylate iAs{sup III} was associated with inhibition of DMAs production by moderate concentrations of iAs{sup III} and with retention of iAs and MAs in cells. No significant correlations were found between the rate of iAs methylation and the thioredoxin reductase activity or glutathione concentration, two factors that modulate the activity of recombinant As3mt. No associations between the rates of iAs methylation and As3mt protein structures were found for the six species examined. Immunoblot analyses indicate that the superior arsenic methylation capacities of dog, rat and monkey hepatocytes examined in this study may be associated with a higher As3mt expression. However, factors other than As3mt expression may also contribute to

  18. Neoplastic cells obtained from Hodgkin's disease are potent stimulators of human primary mixed lymphocyte cultures.

    PubMed

    Fisher, R I; Bostick-Bruton, F; Sauder, D N; Scala, G; Diehl, V

    1983-06-01

    Neoplastic cells obtained from the pleural effusion of a patient with Hodgkin's disease have been maintained in culture since 1978. These tumor cells have been shown to have the cytologic features, cytochemical staining, and cell surface markers of Reed-Sternberg cells. In this study we demonstrate that the cell line termed L428 is a potent stimulator of the primary human mixed lymphocyte reaction. Significant proliferation occurred when mononuclear leukocytes obtained from normal donors were stimulated with radiated L428 cells at responder:stimulator ratios varying from 200:1 to 20:1. Proliferative responses occurred between days 3 and 6 of the cultures with maximal proliferation on day 5. Under optimal culture conditions, mean net proliferative response of 14 normal donors was 51,000 +/- 10,600 dpm. The mixed lymphocyte response was totally blocked by concentrations of monoclonal anti-Ia antibody that had no effect on concanavalin A-induced proliferation. However, the mixed lymphocyte response was not blocked by an anti-K562 cell monoclonal antibody of the same immunoglobulin subclass that binds to the L428 cells. Antigen processing by responder monocytes or Ia-positive cells was not required for the MLC. When responder T cells from two normals were depleted of Ia-bearing cells and monocytes, the mixed lymphocyte reaction between the two normals was eliminated, yet the stimulation of each normal by the L428 cells was not reduced. The cells that proliferated in response to stimulation by the L428 cells were T cells, primarily of the helper subset. No IL 1 activity could be detected in concentrated supernatants of L428 cultures after stimulation of L428 cells by mitogens, phorbol esters, or muramyl dipeptide, or in the MLC. All of these cultures contain fetal calf serum. However, the L428 cells are capable of producing IL 1, because IL 1 was detected when the L428 cells were stimulated with LPS in the absence of fetal calf serum. These neoplastic cells, obtained

  19. Accumulation of pyrethroid compounds in primary cultures of rat cortical neurons

    EPA Science Inventory

    Recent studies have demonstrated that lipophilic compounds (e.g. methylmercury, polychlorinated biphenyls (PCBs) and polybrominated diphenylethers (PBDEs)) rapidly accumulate in cells in culture to concentrations much higher than in the surrounding media. Primary cultures of neur...

  20. Ultrasonic differentiation of normal versus malignant breast epithelial cells in monolayer cultures

    PubMed Central

    Doyle, Timothy E.; Goodrich, Jeffrey B.; Ambrose, Brady J.; Patel, Hemang; Kwon, Soonjo; Pearson, Lee H.

    2010-01-01

    Normal and malignant mammary epithelial cells were studied using laboratory measurements, wavelet analysis, and numerical simulations of monolayer cell cultures to determine whether microscopic breast cancer can be detected in vitro with high-frequency ultrasound. Pulse-echo waveforms were acquired by immersing a broadband, unfocused 50-MHz transducer in the growth media of cell culture well plates and collecting the first reflection from the well bottoms. The simulations included a multilayer pulse-reflection model and a model of two-dimensional arrays of spherical cells and nuclei. The results show that normal and malignant cells produce time-domain signals and spectral features that are significantly different. PMID:21110531

  1. Mitochondrial-dependent manganese neurotoxicity in rat primary astrocyte cultures

    PubMed Central

    Yin, Zhaoobao; Aschner, Judy L.; Santos, Ana Paula dos; Aschner, Michael

    2008-01-01

    Chronic exposure to excessive levels of Mn results in a movement disorder termed manganism, which resembles Parkinson’s disease (PD). The pathogenic mechanisms underlying this disorder are not fully understood. Several lines of evidence implicate astrocytes as an early target of Mn neurotoxicity. In the present study, we investigated the effects of Mn on mitochondrial function. Primary astrocyte cultures were prepared from cerebral cortices of one-day-old Sprague–Dawley rats. We have examined the cellular toxicity of Mn and its effects on the phosphorylation of extracellular signal-regulated kinase (ERK) and activation of the precursor protein of caspase-3. The potentiometric dye, tetramethylrhodamine ethyl ester (TMRE), was used to assess the effect of Mn on astrocytic mitochondrial inner membrane potential (ΔΨm). Our studies show that, in a concentration-dependent manner, Mn induces significant (p<0.05) activation of astrocyte caspase-3 and phosphorylated extracellular signal-regulated kinase (p-ERK). Mn treatment (1 and 6 hrs) also significantly (p<0.01) dissipates the ΔΨm in astrocytes as evidenced by a decrease in mitochondrial TMRE fluorescence. These results suggest that activations of astrocytic caspase-3 and ERK are involved in Mn-induced neurotoxicity via mitochondrial-dependent pathways. PMID:18313649

  2. Aniline Induces Oxidative Stress and Apoptosis of Primary Cultured Hepatocytes.

    PubMed

    Wang, Yue; Gao, Hong; Na, Xiao-Lin; Dong, Shu-Ying; Dong, Hong-Wei; Yu, Jia; Jia, Li; Wu, Yong-Hui

    2016-11-30

    The toxicity and carcinogenicity of aniline in humans and animals have been well documented. However, the molecular mechanism involved in aniline-induced liver toxicity and carcinogenesis remains unclear. In our research, primary cultured hepatocytes were exposed to aniline (0, 1.25, 2.50, 5.0 and 10.0 μg/mL) for 24 h in the presence or absence of N-acetyl-l-cysteine (NAC). Levels of reactive oxygen species (ROS), malondialdehyde (MDA), and glutathione (GSH), activities of superoxide dismutase (SOD) and catalase (CAT), mitochondrial membrane potential, DNA damage, cell viability, and apoptosis were detected. Levels of ROS and MDA were significantly increased and levels of GSH and CAT, activity of SOD, and mitochondrial membrane potential in hepatocytes were significantly decreased by aniline compared with the negative control group. The tail moment and DNA content of the tail in exposed groups were significantly higher than those in the negative control group. Cell viability was reduced and apoptotic death was induced by aniline in a concentration-dependent manner. The phenomena of ROS generation, oxidative damage, loss of mitochondrial membrane potential, DNA damage and apoptosis could be prevented if ROS inhibitor NAC was added. ROS generation is involved in the loss of mitochondrial membrane potential and DNA injury, which may play a role in aniline-induced apoptosis in hepatocytes. Our study provides insight into the mechanism of aniline-induced toxicity and apoptosis of hepatocytes.

  3. Transport Mechanism of Nicotine in Primary Cultured Alveolar Epithelial Cells.

    PubMed

    Takano, Mikihisa; Nagahiro, Machi; Yumoto, Ryoko

    2016-02-01

    Nicotine is absorbed from the lungs into the systemic circulation during cigarette smoking. However, there is little information concerning the transport mechanism of nicotine in alveolar epithelial cells. In this study, we characterized the uptake of nicotine in rat primary cultured type II (TII) and transdifferentiated type I-like (TIL) epithelial cells. In both TIL and TII cells, [(3)H]nicotine uptake was time and temperature-dependent, and showed saturation kinetics. [(3)H]Nicotine uptake in these cells was not affected by Na(+), but was sensitive to extracellular and intracellular pH, suggesting the involvement of a nicotine/proton antiport system. The uptake of [(3)H]nicotine in these cells was potently inhibited by organic cations such as clonidine, diphenhydramine, and pyrilamine, but was not affected by substrates and/or inhibitors of known organic cation transporters such as carnitine, 1-methyl-4-phenylpyridinium, and tetraethylammonium. In addition, the uptake of [(3)H]nicotine in TIL cells was stimulated by preloading the cells with unlabeled nicotine, pyrilamine, and diphenhydramine, but not with tetraethylammonium. These results suggest that a novel proton-coupled antiporter is involved in the uptake of nicotine in alveolar epithelial cells and its absorption from the lungs into the systemic circulation.

  4. Aniline Induces Oxidative Stress and Apoptosis of Primary Cultured Hepatocytes

    PubMed Central

    Wang, Yue; Gao, Hong; Na, Xiao-Lin; Dong, Shu-Ying; Dong, Hong-Wei; Yu, Jia; Jia, Li; Wu, Yong-Hui

    2016-01-01

    The toxicity and carcinogenicity of aniline in humans and animals have been well documented. However, the molecular mechanism involved in aniline-induced liver toxicity and carcinogenesis remains unclear. In our research, primary cultured hepatocytes were exposed to aniline (0, 1.25, 2.50, 5.0 and 10.0 μg/mL) for 24 h in the presence or absence of N-acetyl-l-cysteine (NAC). Levels of reactive oxygen species (ROS), malondialdehyde (MDA), and glutathione (GSH), activities of superoxide dismutase (SOD) and catalase (CAT), mitochondrial membrane potential, DNA damage, cell viability, and apoptosis were detected. Levels of ROS and MDA were significantly increased and levels of GSH and CAT, activity of SOD, and mitochondrial membrane potential in hepatocytes were significantly decreased by aniline compared with the negative control group. The tail moment and DNA content of the tail in exposed groups were significantly higher than those in the negative control group. Cell viability was reduced and apoptotic death was induced by aniline in a concentration-dependent manner. The phenomena of ROS generation, oxidative damage, loss of mitochondrial membrane potential, DNA damage and apoptosis could be prevented if ROS inhibitor NAC was added. ROS generation is involved in the loss of mitochondrial membrane potential and DNA injury, which may play a role in aniline-induced apoptosis in hepatocytes. Our study provides insight into the mechanism of aniline-induced toxicity and apoptosis of hepatocytes. PMID:27916916

  5. Measurement of Primary Ejecta From Normal Incident Hypervelocity Impact on Lunar Regolith Simulant

    NASA Technical Reports Server (NTRS)

    Edwards, David L.; Cooke, William; Moser, Danielle; Swift, Wesley

    2007-01-01

    The National Aeronautics and Space Administration (NASA) continues to make progress toward long-term lunar habitation. Critical to the design of a lunar habitat is an understanding of the lunar surface environment. A subject for further definition is the lunar primary ejecta environment. The document NASA SP-8013 was developed for the Apollo program and is the latest definition of the primary ejecta environment. There is concern that NASA SP-8013 may over-estimate the lunar primary ejecta environment. NASA's Meteoroid Environment Office (MEO) has initiated several tasks to improve the accuracy of our understanding of the lunar surface primary ejecta environment. This paper reports the results of experiments on projectile impact into pumice targets, simulating lunar regolith. The Ames Vertical Gun Range (AVGR) was used to accelerate spherical Pyrex projectiles of 0.29g to velocities ranging between 2.5 km/s and 5.18 km/s. Impact on the pumice target occurred at normal incidence. The ejected particles were detected by thin aluminum foil targets placed around the pumice target in a 0.5 Torr vacuum. A simplistic technique to characterize the ejected particles was formulated. Improvements to this technique will be discussed for implementation in future tests.

  6. Phosphorylation of intracellular proteins related to the multihormonal regulation of prolactin: comparison of normal anterior pituitary cells in culture with the tumor-derived GH cell lines

    SciTech Connect

    Beretta, L.; Boutterin, M.C.; Sobel, A.

    1988-01-01

    We have previously identified a group of cytoplasmic phosphoproteins (proteins 1-11) whose phosphorylation could be related, on a pharmacological basis, to the multihormonal regulation of PRL synthesis and release in the anterior pituitary tumor-derived GH cell lines. Phosphoproteins with identical migration properties on two-dimensional electrophoresis gels were also detectable in normal rat anterior pituitary cells in culture. We designed appropriate culture and (/sup 32/P) phosphate-labeling conditions allowing to analyze the regulation of the phosphorylation of these proteins in normal pituitary cells. TRH, 12-O-tetradecanoylphorbol-13-acetate, and vasoactive intestinal peptide induced the same qualitative changes in phosphorylation of proteins 1-11 in normal as in GH cells. Quantitative differences observed are most likely due to the heterogeneity of primary pituitary cultures. Phosphorylation changes affecting proteins 14-16, not previously detected in GH cells, were also observed with normal anterior pituitary cells. GH cell lines have lost the sensitivity of pituitary lactotrophs for dopamine, an important physiological inhibitor of PRL synthesis and release. In normal anterior pituitary cells in culture, dopamine inhibited also the TRH-stimulated phosphorylation of proteins 1-10, thus strengthening the correlation between phosphorylation of these proteins and multihormonal regulation of pituitary cell functions. Our results indicate: 1) that the same phosphoproteins as in GH cells are related to the multihormonal regulation of nontumoral, normal anterior pituitary cells in culture; 2) that dopamine acts by interfering with the phosphorylation of these proteins.

  7. Regulation of heme metabolism in normal and sideroblastic bone marrow cells in culture

    SciTech Connect

    Ibraham, N.G.; Lutton, J.D.; Hoffman, R.; Levere, R.D.

    1985-05-01

    Heme metabolism was examined in developing in vitro erythroid colonies (CFUE) and in bone marrow samples taken directly from four normal donors and four patients with sideroblastic anemia. Maximum activities of delta-aminolevulinic acid synthase (ALAS), ALA dehydratase (ALAD), and /sup 14/C-ALA incorporation into heme were achieved in normal marrow CFUE after 8 days of culture, whereas heme oxygenase progressively decreased to low levels of activity during the same period. Assays on nucleated bone marrow cells taken directly from patients revealed that ALAS activity was considerably reduced in idiopathic sideroblastic anemia (IASA) and X-linked sideroblastic anemia (X-SA) bone marrow specimens, whereas the activity increased more than twofold (normal levels) when cells were assayed from 8-day CFUE. In all cases, ALAD activity appeared to be within normal levels. Measurement of heme synthesis revealed that normal levels of /sup 14/C-ALA incorporation into heme were achieved in IASA cells but were reduced in X-SA cells. In marked contrast to levels in normal cells, heme oxygenase was found to be significantly elevated (two- to fourfold) in bone marrow cells taken directly from patients with IASA and X-SA. Results from this study demonstrate that IASA and X-SA bone marrow cells have disturbances in ALAS and heme metabolism, and that erythropoiesis (CFUE) can be restored to normal levels when cells are cultured in methylcellulose.

  8. Neuroprotective Effect of Carnosine on Primary Culture of Rat Cerebellar Cells under Oxidative Stress.

    PubMed

    Lopachev, A V; Lopacheva, O M; Abaimov, D A; Koroleva, O V; Vladychenskaya, E A; Erukhimovich, A A; Fedorova, T N

    2016-05-01

    Dipeptide carnosine (β-alanyl-L-histidine) is a natural antioxidant, but its protective effect under oxidative stress induced by neurotoxins is studied insufficiently. In this work, we show the neuroprotective effect of carnosine in primary cultures of rat cerebellar cells under oxidative stress induced by 1 mM 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH), which directly generates free radicals both in the medium and in the cells, and 20 nM rotenone, which increases the amount of intracellular reactive oxygen species (ROS). In both models, adding 2 mM carnosine to the incubation medium decreased cell death calculated using fluorescence microscopy and enhanced cell viability estimated by the MTT assay. The antioxidant effect of carnosine inside cultured cells was demonstrated using the fluorescence probe dichlorofluorescein. Carnosine reduced by half the increase in the number of ROS in neurons induced by 20 nM rotenone. Using iron-induced chemiluminescence, we showed that preincubation of primary neuronal cultures with 2 mM carnosine prevents the decrease in endogenous antioxidant potential of cells induced by 1 mM AAPH and 20 nM rotenone. Using liquid chromatography-mass spectrometry, we showed that a 10-min incubation of neuronal cultures with 2 mM carnosine leads to a 14.5-fold increase in carnosine content in cell lysates. Thus, carnosine is able to penetrate neurons and exerts an antioxidant effect. Western blot analysis revealed the presence of the peptide transporter PEPT2 in rat cerebellar cells, which suggests the possibility of carnosine transport into the cells. At the same time, Western blot analysis showed no carnosine-induced changes in the level of apoptosis regulating proteins of the Bcl-2 family and in the phosphorylation of MAP kinases, which suggests that carnosine could have minimal or no side effects on proliferation and apoptosis control systems in normal cells.

  9. Erythropoietin triggers a burst of GATA-1 in normal human erythroid cells differentiating in tissue culture.

    PubMed Central

    Dalyot, N; Fibach, E; Ronchi, A; Rachmilewitz, E A; Ottolenghi, S; Oppenheim, A

    1993-01-01

    GATA-1 is a central transcription-activator of erythroid differentiation. In the present work we have studied the kinetics of its expression and activity during development of normal human erythroid progenitors, grown in primary cultures. In response to the addition of erythropoietin (Epo), the cells undergo proliferation and differentiation in a synchronized fashion. This recently developed experimental system allows biochemical dissection of erythroid differentiation in a physiological meaningful environment. No DNA-binding activity of GATA-1 could be detected before the addition of Epo, although a very low level of mRNA was observed. Following Epo addition there was a sharp parallel rise in both mRNA and DNA-binding activity, consistent with positive autoregulation of the GATA-1 gene. After reaching a peak on day 7-9, both mRNA and protein activity decreased. The binding activity of the ubiquitous factor SP1 showed a biphasic pattern; its second peak usually coincided with the GATA-1 peak, suggesting that SP1 also plays a specific role in erythroid maturation. The highest activity of GATA-1 per erythroid cell was found on day 6-8, immediately preceding the major rise in globin gene mRNA and in the number of hemoglobinized cells. The results imply that a high level of GATA-1 activity is necessary for globin gene expression and erythroid maturation, suggesting that a requirement for a threshold concentration of GATA-1 is part of the mechanism that determines the final steps of erythroid maturation. Images PMID:8371977

  10. Low calcium culture condition induces mesenchymal cell-like phenotype in normal human epidermal keratinocytes

    SciTech Connect

    Takagi, Ryo; Yamato, Masayuki; Murakami, Daisuke; Sugiyama, Hiroaki; Okano, Teruo

    2011-08-26

    Highlights: {yields} Normal human epidermal keratinocytes serially cultured under low calcium concentration were cytokeratin and vimentin double positive cells. {yields} The human keratinocytes expressed some epithelial stem/progenitor cell makers, mesenchymal cell markers, and markers of epithelial-mesenchymal transition. {yields} Mesenchymal cell-like phenotype in the keratinocytes was suppressed under high-calcium condition. -- Abstract: Epithelial-mesenchymal transition (EMT) is an important cellular phenomenon in organ developments, cancer invasions, and wound healing, and many types of transformed cell lines are used for investigating for molecular mechanisms of EMT. However, there are few reports for EMT in normal human epithelial cells, which are non-transformed or non-immortalized cells, in vitro. Therefore, normal human epidermal keratinocytes (NHEK) serially cultured in low-calcium concentration medium (LCM) were used for investigating relations between differentiation and proliferation and mesenchymal-like phenotype in the present study, since long-term cultivation of NHEK is achieved in LCM. Interestingly, NHEK serially cultured in LCM consisted essentially of cytokeratin-vimentin double positive cells (98%), although the NHEK exhibited differentiation under high-calcium culture condition with 3T3 feeder layer. The vimentin expression was suppressed under high-calcium condition. These results may indicate the importance of mesenchymal-like phenotype for serially cultivation of NHEK in vitro.

  11. Exploring Culture in Locally Published English Textbooks for Primary Education in Turkey

    ERIC Educational Resources Information Center

    Kirkgöz, Yasemin; Agçam, Reyhan

    2011-01-01

    Since language and culture are closely interwoven, the integration of culture into textbooks used for teaching English as a second/foreign language has become a widely accepted phenomenon. This study investigates the cultural elements in locally published English textbooks used for Turkish primary schools following two major curriculum innovations…

  12. Effects of nutritional and hormonal factors on the metabolism of retinol-binding protein by primary cultures of rat hepatocytes

    SciTech Connect

    Dixon, J.L.; Goodman, D.S.

    1987-01-01

    Studies were conducted to explore hormonal and nutritional factors that might be involved in the regulation of retinol-binding protein (RBP) synthesis and secretion by the liver. The studies employed primary cultures of hepatocytes from normal rats. When cells were cultured in Dulbecco's modified Eagle's medium alone, a high rate of RBP secretion was observed initially, which declined and became quite low by 24 hr. Supplementing the medium with amino acids maintained RBP and albumin secretion at moderate (but less than initial) rates for at least 3 days. Further addition of dexamethasone maintained the production and secretion rates of RBP, transthyretin, and albumin close to the initial rates for up to 3-5 days in culture as measured by radioimmunoassay. Hormonally treated hepatocytes produced and secreted RBP, transthyretin, and albumin at both absolute and relative rates similar to physiological values, as estimated from rates reported by others from studies in vivo and with perfused livers. Glucagon addition partially maintained the secretion rates of these 3 proteins, but less effectively than did dexamethasone. A number of other hormones, added singly or in combination, did not affect RBP production or secretion. Addition of retinol to the cultured normal hepatocytes was without effect upon RBP secretion. These studies show that supplementing the culture medium of hepatocytes with amino acids and dexamethasone maintains RBP production and secretion for several days. In normal hepatocytes, with ample supply of retinol available within the cell, addition of exogenous retinol does not appear to influence RBP metabolism or secretion by the cells.

  13. Effect of lectin from Chelidonium majus L. on normal and cancer cells in culture.

    PubMed

    Fik, E; Wołuń-Cholewa, M; Kistowska, M; Warchoł, J B; Goździcka-Józefiak, A

    2001-01-01

    Lectin from Chelidonium majus L. (CML) significantly stimulates the proliferation of human lymphocytes and has hemagglutination activity towards group B human erythrocytes and potent antimicrobial properties against multiresistant enterococci and staphylococci. In the present work we describe the effect of lectin from Chelidonium majus L on normal and cancercells in culture in vitro. The studies were performed on three types of cells: CHO, R2C and on normal mouse fibroblasts. Effects on the cultures were examined 24 h after addition of CML. Exposure to CML resulted in growth inhibition of CHO and R2C cells but not of fibroblasts. Moreover, evident apoptotic lesions were observed in CHO cells and less well marked apoptotic lesions in R2C cells. In contrast, only insignificant numbers of fibroblasts reacted to the applied lectin.

  14. Cultural democracy: the way forward for primary care of hard to reach New Zealanders.

    PubMed

    Finau, Sitaleki A; Finau, Eseta

    2007-09-01

    The use of cultural democracy, the freedom to practice one's culture without fear, as a framework for primary care service provision is essential for improved health service in a multi cultural society like New Zealand. It is an effective approach to attaining health equity for all. Many successful health ventures are ethnic specific and have gone past cultural competency to the practice of cultural democracy. That is, the services are freely taking on the realities of clients without and malice from those of other ethnicities. In New Zealand the scientific health service to improve the health of a multi cultural society are available but there is a need to improve access and utilization by hard to reach New Zealanders. This paper discusses cultural democracy and provide example of how successful health ventures that had embraced cultural democracy were implemented. It suggests that cultural democracy will provide the intellectual impetus and robust philosophy for moving from equality to equity in health service access and utilization. This paper would provide a way forward to improved primary care utilization, efficiency, effectiveness and equitable access especially for the hard to reach populations. use the realities of Pacificans in New Zealand illustrate the use of cultural democracy, and thus equity to address the "inverse care law" of New Zealand. The desire is for primary care providers to take cognizance and use cultural democracy and equity as the basis for the design and practice of primary health care for the hard to reach New Zealanders.

  15. Stored erythrocytes have less capacity than normal erythrocytes to support primary haemostasis.

    PubMed

    Reinhart, Walter H; Zehnder, Linda; Schulzki, Thomas

    2009-04-01

    Primary haemostasis is mediated by platelet aggregation. Red blood cells (RBCs) are involved in this process. We hypothesised that stored RBCs could have less capacity to support primary haemostasis. This was tested with RBC units from 17 healthy volunteers stored for 45 days. Fresh citrated blood was taken again from the same donors and platelet-rich plasma was prepared, in which RBCs were resuspended with a constant haematocrit (40%), but changing fractions of stored versus fresh autologous RBCs (0, 25, 50, 75, and 100%, respectively). A platelet function analyser PFA-100((R)) was used. In this instrument blood is aspirated through a membrane pore coated with collagen and either epinephrine (EPI) or ADP, which causes platelets to adhere, aggregate, and form an occluding plug, which stops blood flow and is measured as closure time (CT). We found that the CT increased with increasing fractions of stored blood. CT-EPI was 121 +/- 17 seconds [s], 129 +/- 32 s, 164 +/- 45 s (p < 0.000, ANOVA), 214 +/- 54 s (p < 0.0001), and 273 +/- 36 s (p < 0.0001) for 0, 25, 50, 75, and 100% stored RBCs. For CT-ADP the values were 91 +/- 22 s, 95 +/- 12 s, 101 +/- 13 s, 124 +/- 44 s (p = 0.004), and 191 +/- 72 s (p < 0.0001), respectively. We conclude that stored RBCs have less capacity than normal RBCs to support primary haemostasis by platelet aggregation in vitro, suggesting a decreased capacity of stored RBCs to bring platelets into close contact with the wall, which may contribute to sustained bleeding seen after mass transfusion.

  16. L-carnosine reduces telomere damage and shortening rate in cultured normal fibroblasts.

    PubMed

    Shao, Lan; Li, Qing-Huan; Tan, Zheng

    2004-11-12

    Telomere is the repetitive DNA sequence at the end of chromosomes, which shortens progressively with cell division and limits the replicative potential of normal human somatic cells. L-carnosine, a naturally occurring dipeptide, has been reported to delay the replicative senescence, and extend the lifespan of cultured human diploid fibroblasts. In this work, we studied the effect of carnosine on the telomeric DNA of cultured human fetal lung fibroblast cells. Cells continuously grown in 20 mM carnosine exhibited a slower telomere shortening rate and extended lifespan in population doublings. When kept in a long-term nonproliferating state, they accumulated much less damages in the telomeric DNA when cultured in the presence of carnosine. We suggest that the reduction in telomere shortening rate and damages in telomeric DNA made an important contribution to the life-extension effect of carnosine.

  17. Human uracil DNA N-glycosidase: studies in normal and repair defective cultured fibroblasts.

    PubMed Central

    Kuhnlein, U; Lee, B; Linn, S

    1978-01-01

    Uracil DNA N-glycosidase, an enzyme which participates in the excision of uracil from DNA, was measured in extracts from fibroblasts lines cultured from normal subjects, from several subjects with the genetic disease xeroderma pigmentosum, and from a subject with ataxia telangiectasia. The cell lines representative of complementation groups A and D of xeroderma pigmentosum and of ataxia telangiectasia had roughly the same level of activity as did the normal cells. On the other hand, cells from two xeroderma pigmentosum variants (XP4BE and XP13BE) had roughly half the normal level of activity, and cells from the heterozygous mother of XP4BE had an intermediate level of activity. In spite of these quantitative differences, no systematic alterations in reaction characteristics, apparent Km for substrate, or purification characteristics were noted for enzyme from any of the lines. Thus a causal relationship, if any, between levels of activity and the disease symptoms is equivocal. PMID:643602

  18. Subcellular compartmentalization of saccharide moieties in cultured normal and malignant cells

    PubMed Central

    1980-01-01

    We studied subcellular localization of saccharide moieties in cultured normal and malignant cells fixed in paraformaldehyde and treated with a nonionic detergent, using lectins specific for various surgar residues as probes in fluorescence microscopy. In normal cells, concanavalin A and Lens culinaris agglutinin, specific for mannose-rich carbohydrate cores in glycoproteins, labeled the endoplasmic reticulum as a wide perinuclear region. Other lectins, on the other hand, stained the Golgi apparatus as a juxtanuclear reticular structure. A similar compartmentalization was also seen in all malignant cells studied, although the Golgi apparatus in these cells was distinctly vesicular in appearance. Our results indicate that saccharide moieties in both normal and malignant cells are similarly compartmentalized, and thus speak in favor of a unidirectional subcellular flow for both membrane and secreted glycoconjugates. PMID:7372714

  19. Subcellular compartmentalization of saccharide moieties in cultured normal and malignant cells.

    PubMed

    Virtanen, I; Ekblom, P; Laurila, P

    1980-05-01

    We studied subcellular localization of saccharide moieties in cultured normal and malignant cells fixed in paraformaldehyde and treated with a nonionic detergent, using lectins specific for various surgar residues as probes in fluorescence microscopy. In normal cells, concanavalin A and Lens culinaris agglutinin, specific for mannose-rich carbohydrate cores in glycoproteins, labeled the endoplasmic reticulum as a wide perinuclear region. Other lectins, on the other hand, stained the Golgi apparatus as a juxtanuclear reticular structure. A similar compartmentalization was also seen in all malignant cells studied, although the Golgi apparatus in these cells was distinctly vesicular in appearance. Our results indicate that saccharide moieties in both normal and malignant cells are similarly compartmentalized, and thus speak in favor of a unidirectional subcellular flow for both membrane and secreted glycoconjugates.

  20. Calcium currents and transients in co-cultured contracting normal and Duchenne muscular dystrophy human myotubes

    PubMed Central

    Imbert, Nathalie; Vandebrouck, Clarisse; Duport, Gérard; Raymond, Guy; Hassoni, Abdul A; Constantin, Bruno; Cullen, Michael J; Cognard, Christian

    2001-01-01

    The goal of the present study was to investigate differences in calcium movements between normal and Duchenne muscular dystrophy (DMD) human contracting myotubes co-cultured with explants of rat spinal cord with attached dorsal root ganglia. Membrane potential, variations of intracellular calcium concentration and T- and L-type calcium currents were recorded. Further, a descriptive and quantitative study by electron microscopy of the ultrastructure of the co-cultures was carried out. The resting membrane potential was slightly less negative in DMD (−61.4 ± 1.1 mV) than in normal myotubes (−65.5 ± 0.9 mV). Both types of myotube displayed spontaneous action potentials (mean firing frequency, 0.42 and 0.16 Hz, respectively), which triggered spontaneous calcium transients measured with Indo-1. The time integral under the spontaneous Ca2+ transients was significantly greater in DMD myotubes (97 ± 8 nm s) than in normal myotubes (67 ± 13 nm s). The L- and T-type current densities estimated from patch-clamp recordings were smaller in DMD cells (2.0 ± 0.5 and 0.90 ± 0.19 pA pF−1, respectively) than in normal cells (3.9 ± 0.7 and 1.39 ± 0.30 pA pF−1, respectively). The voltage-dependent inactivation relationships revealed a shift in the conditioning potential at which inactivation is half-maximal (Vh,0.5) of the T- and L-type currents towards less negative potentials, from −72.1 ± 0.7 and −53.7 ± 1.5 mV in normal cells to −61.9 ± 1.4 and −29.2 ± 1.4 mV in DMD cells, respectively. Both descriptive and quantitative studies by electron microscopy suggested a more advanced development of DMD myotubes as compared to normal ones. This conclusion was supported by the significantly larger capacitance of the DMD myotubes (408 ± 45 pF) than of the normal myotubes (299 ± 34 pF) of the same apparent size. Taken together, these results show that differences in T- and L-type calcium currents between normal and DMD myotubes cannot simply explain all observed

  1. Production of Normal Mammalian Organ Culture Using a Medium Containing Mem-Alpha, Leibovitz L 15, Glucose Galactose Fructose

    NASA Technical Reports Server (NTRS)

    Goodwin, Thomas J. (Inventor); Wolf, David A. (Inventor); Spaulding, Glenn F. (Inventor); Prewett, Tacey L. (Inventor)

    1999-01-01

    Normal mammalian tissue and the culturing process has been developed for the three groups of organ, structural and blood tissue. The cells are grown in vitro under micro- gravity culture conditions and form three dimensional cells aggregates with normal cell function. The microgravity culture conditions may be microgravity or simulated microgravity created in a horizontal rotating wall culture vessel. The medium used for culturing the cells, especially a mixture of epithelial and mesenchymal cells contains a mixture of Mem-alpha and Leibovits L15 supplemented with glucose, galactose and fructose.

  2. Transformation of adult rat cardiac myocytes in primary culture.

    PubMed

    Banyasz, Tamas; Lozinskiy, Ilya; Payne, Charles E; Edelmann, Stephanie; Norton, Byron; Chen, Biyi; Chen-Izu, Ye; Izu, Leighton T; Balke, C William

    2008-03-01

    We characterized the morphological, electrical and mechanical alterations of cardiomyocytes in long-term cell culture. Morphometric parameters, sarcomere length, T-tubule density, cell capacitance, L-type calcium current (I(Ca,L)), inward rectifier potassium current (I(K1)), cytosolic calcium transients, action potential and contractile parameters of adult rat ventricular myocytes were determined on each day of 5 days in culture. We also analysed the health of the myocytes using an apoptotic/necrotic viability assay. The data show that myocytes undergo profound morphological and functional changes during culture. We observed a progressive reduction in the cell area (from 2502 +/- 70 microm(2) on day 0 to 1432 +/- 50 microm(2) on day 5), T-tubule density, systolic shortening (from 0.11 +/- 0.02 to 0.05 +/- 0.01 microm) and amplitude of calcium transients (from 1.54 +/- 0.19 to 0.67 +/- 0.19) over 5 days of culture. The negative force-frequency relationship, characteristic of rat myocardium, was maintained during the first 2 days but diminished thereafter. Cell capacitance (from 156 +/- 8 to 105 +/- 11 pF) and membrane currents were also reduced (I(Ca,L), from 3.98 +/- 0.39 to 2.12 +/- 0.37 pA pF; and I(K1), from 34.34p +/- 2.31 to 18.00 +/- 5.97 pA pF(-1)). We observed progressive depolarization of the resting membrane potential during culture (from 77.3 +/- 2.5 to 34.2 +/- 5.9 mV) and, consequently, action potential morphology was profoundly altered as well. The results of the viability assays indicate that these alterations could not be attributed to either apoptosis or necrosis but are rather an adaptation to the culture conditions over time.

  3. An Air-Liquid Interface Culture System for 3D Organoid Culture of Diverse Primary Gastrointestinal Tissues.

    PubMed

    Li, Xingnan; Ootani, Akifumi; Kuo, Calvin

    2016-01-01

    Conventional in vitro analysis of gastrointestinal epithelium usually relies on two-dimensional (2D) culture of epithelial cell lines as monolayer on impermeable surfaces. However, the lack of context of differentiation and tissue architecture in 2D culture can hinder the faithful recapitulation of the phenotypic and morphological characteristics of native epithelium. Here, we describe a robust long-term three-dimensional (3D) culture methodology for gastrointestinal culture, which incorporates both epithelial and mesenchymal/stromal components into a collagen-based air-liquid interface 3D culture system. This system allows vigorously expansion of primary gastrointestinal epithelium for over 60 days as organoids with both proliferation and multilineage differentiation, indicating successful long-term intestinal culture within a microenvironment accurately recapitulating the stem cell niche.

  4. The rough endoplasmic reticulum and the Golgi apparatus visualized using specific antibodies in normal and tumoral prolactin cells in culture

    PubMed Central

    1983-01-01

    Antibodies directed against membrane components of dog pancreas rough endoplasmic reticulum (A-RER) and rat liver Golgi apparatus (A-Golgi) (Louvard, D., H. Reggio, and G. Warren, 1982, J. Cell Biol. 92:92-107) have been applied to cultured rat prolactin (PRL) cells, either normal cells in primary cultures, or clonal GH3 cells. In normal PRL cells, the A-RER stained the membranes of the perinuclear cisternae as well as those of many parallel RER cisternae. The A-Golgi stained part of the Golgi membranes. In the stacks it stained the medial saccules and, with a decreasing intensity, the saccules of the trans side, as well as, in some cells, a linear cisterna in the center of the Golgi zone. It also stained the membrane of many small vesicles as well as that of lysosomelike structures in all cells. In contrast, it never stained the secretory granule membrane, except at the level of very few segregating granules on the trans face of the Golgi zone. In GH3 cells the A-RER stained the membrane of the perinuclear cisternae, as well as that of short discontinuous flat cisternae. The A-Golgi stained the same components of the Golgi zone as in normal PRL cells. In some cells of both types the A-Golgi also stained discontinuous patches on the plasma membrane and small vesicles fusing with the plasma membrane. Immunostaining of Golgi membranes revealed modifications of membrane flow in relation to either acute stimulation of PRL release by thyroliberin or inhibition of basal secretion by monensin. PMID:6404908

  5. Establishing and maintaining primary cell cultures derived from the ctenophore Mnemiopsis leidyi.

    PubMed

    Vandepas, Lauren E; Warren, Kaitlyn J; Amemiya, Chris T; Browne, William E

    2017-04-01

    We have developed an efficient method for the preparation and maintenance of primary cell cultures isolated from adult Mnemiopsis leidyi, a lobate ctenophore. Our primary cell cultures are derived from tissue explants or enzymatically dissociated cells, and maintained in a complex undefined ctenophore mesogleal serum. These methods can be used to isolate, maintain and visually monitor ctenophore cells to assess proliferation, cellular morphology and cell differentiation in future studies. Exemplar cell types that can be easily isolated from primary cultures include proliferative ectodermal and endodermal cells, motile amebocyte-like cells, and giant smooth muscle cells that exhibit inducible contractile properties. We have also derived 'tissue envelopes' containing sections of endodermal canal surrounded by mesoglea and ectoderm that can be used to monitor targeted cell types in an in vivo context. Access to efficient and reliably generated primary cell cultures will facilitate the analysis of ctenophore development, physiology and morphology from a cell biological perspective.

  6. Pyrethroid insecticide accumulation in primary cultures of cortical neurons in vitro

    EPA Science Inventory

    Primary cultures of neurons have been widely utilized to study the actions of pyrethroids and other neurotoxicants, with the presumption that the media concentration accurately reflects the dose received by the cells. However, recent studies have demonstrated that lipophilic comp...

  7. Toward a world-class culture in primary care: listening to the voice of the customer.

    PubMed

    Benson, D S

    1996-01-01

    This article on creating a world-class culture in primary care stresses the importance of customers. The concept of patients as external customers is addressed; the emphasis, however, is on healthcare professionals' responsibilities toward one another as internal customers.

  8. Homophobia, Transphobia and Culture: Deconstructing Heteronormativity in English Primary Schools

    ERIC Educational Resources Information Center

    DePalma, Renee; Jennett, Mark

    2010-01-01

    This article presents some of the advances in legal support for addressing homophobia and transphobia in school settings and provides a critique of school-based policies that focus on these phenomena as particular incidents involving bullies and victims. Defining heteronormativity as a cultural phenomenon underpinning recognisable acts of…

  9. Gene expression analysis of primary normal human hepatocytes infected with human hepatitis B virus

    PubMed Central

    Ryu, Hyun Mi; Park, Sung Gyoo; Yea, Sung Su; Jang, Won Hee; Yang, Young-Il; Jung, Guhung

    2006-01-01

    AIM: To find the relationship between hepatitis B virus (HBV) and hepatocytes during the initial state of infection by cDNA microarray. METHODS: Primary normal human hepatocytes (PNHHs) were isolated and infected with HBV. From the PNHHs, RNA was isolated and inverted into complement DNA (cDNA) with Cy3- or Cy5- labeled dUTP for microarray analysis. The labeled cDNA was hybridized with microarray chip, including 4224 cDNAs. From the image of the microarray, expression profiles were produced and some of them were confirmed by RT-PCR, immunoblot analysis, and NF-κB luciferase reporter assay. RESULTS: From the cDNA microarray, we obtained 98 differentially regulated genes. Of the 98 genes, 53 were up regulated and 45 down regulated. Interestingly, in the up regulated genes, we found the TNF signaling pathway-related genes: LT-α, TRAF2, and NIK. By using RT-PCR, we confirmed the up-regulation of these genes in HepG2, Huh7, and Chang liver cells, which were transfected with pHBV1.2×, a plasmid encoding all HBV messages. Moreover, these three genes participated in HBV-mediated NF-κB activation. CONCLUSION: During the initial state of HBV infection, hepatocytes facilitate the activation of NF-κB through up regulation of LT-α, TRAF2, and NIK. PMID:16937494

  10. Family, Culture and Achievement in the Primary Schools of Botswana.

    ERIC Educational Resources Information Center

    Lynch, Patrick D.

    This paper traces the development of the educational system of Botswana, a small south African country of one million, emphasizing its democratic origins and customs, historical influences, social trends, and economic support. Changes in the educational system, especially in universal primary education, since its independence from Great Britain in…

  11. English and French Pedagogical Cultures: Convergence and Divergence in Cameroonian Primary School Teachers' Discourse

    ERIC Educational Resources Information Center

    Esch, Edith

    2012-01-01

    This article approaches the phenomenon of the continuing influence of French and English pedagogical cultures in Africa relying on post-modern notions of time and space. It reports on a project carried out in Cameroon where both cultures are in contact and where the teachers from two primary schools were observed and interviewed over a period of…

  12. Contribution to Cultural Organization, Working Motivation and Job Satisfaction on the Performance of Primary School Teacher

    ERIC Educational Resources Information Center

    Murtedjo; Suharningsih

    2016-01-01

    The purposes of this study are: (1) describes the performance of the teacher, organizational culture, work motivation and job satisfaction; (2) determine whether there is a significant direct relationship between organizational culture, work motivation and job satisfaction on the performance of primary school teachers. Through the study of the…

  13. Turkish Primary School Teachers' Perceptions of School Culture Regarding ICT Integration

    ERIC Educational Resources Information Center

    Tezci, Erdogan

    2011-01-01

    The current study aimed at identifying Turkish primary school teachers' perceptions of school culture regarding ICT integration in education. In addition, the current study was designed to investigate factors that might influence their perceptions. The participants were 1540 primary school teachers. The findings revealed that the teachers'…

  14. The Effect of Organizational Trust on the Culture of Teacher Leadership in Primary Schools

    ERIC Educational Resources Information Center

    Demir, Kamile

    2015-01-01

    The purpose of this research is to examine the effect of the level of trust of primary school teachers towards their organization in relation to their perceptions of the school having a culture of teacher leadership. Participants of the study consisted of 378 teachers working in Burdur public primary schools. The data collection tool used two…

  15. Meaningful Cultural Learning by Imitative Participation: The Case of Abstract Thinking in Primary School

    ERIC Educational Resources Information Center

    van Oers, Bert

    2012-01-01

    The article describes a theory-driven approach to meaningful learning in primary schools, based on the Vygotskian cultural-historical theory of human development and learning. This approach is elaborated into an educational concept called "developmental education" that is implemented in the Netherlands in many primary schools. In this…

  16. A qualitative study of the cultural changes in primary care organisations needed to implement clinical governance.

    PubMed Central

    Marshall, Martin; Sheaff, Rod; Rogers, Anne; Campbell, Stephen; Halliwell, Shirley; Pickard, Susan; Sibbald, Bonnie; Roland, Martin

    2002-01-01

    BACKGROUND: It is commony claimed that changing the culture of health organisations is a fundamental prerequisite for improving the National Health Service (NHS). Little is currently known about the nature or importance of culture and cultural change in primary care groups and trusts (PCG/Ts) or their constituent general practices. AIMS: To investigate the importance of culture and cultural change for the implementation of clinical governance in general practice by PCG/Ts, to identify perceived desirable and undesirable cultural attributes of general practice, and to describe potential facilitators and barriers to changing culture. DESIGN: Qualitative: case studies using data derived from semi-structured interviews and review of documentary evidence. SETTING: Fifty senior non-clinical and clinical managers from 12 purposely sampled PCGs or trusts in England. RESULTS: Senior primary care managers regard culture and cultural change as fundamental aspects of clinical governance. The most important desirable cultural traits were the value placed on a commitment to public accountability by the practices, their willingness to work together and learn from each other, and the ability to be self-critical and learn from mistakes. The main barriers to cultural change were the high level of autonomy of practices and the perceived pressure to deliver rapid measurable changes in general practice. CONCLUSIONS: The culture of general practice is perceived to be an important component of health system reform and quality improvement. This study develops our understanding of a changing organisational culture in primary care; however, further work is required to determine whether culture is a useful practical lever for initiating or managing improvement. PMID:12171222

  17. Establishment of a Novel Model for Anticancer Drug Resistance in Three-Dimensional Primary Culture of Tumor Microenvironment

    PubMed Central

    Sakurai, Masashi; Enjoji, Shuhei; Umata, Koji; Fujiwara, Nobuyuki; Tsuji, Shunya; Hazama, Shoichi

    2016-01-01

    Tumor microenvironment has been implicated in tumor development and progression. As a three-dimensional tumor microenvironment model, air liquid interface (ALI) organoid culture from oncogene transgenic mouse gastrointestinal tissues was recently produced. However, ALI organoid culture system from tissues of colorectal cancer patients has not been established. Here, we developed an ALI organoid model from normal and tumor colorectal tissues of human patients. Both organoids were successfully generated and showed cystic structures containing an epithelial layer and surrounding mesenchymal stromal cells. Structures of tumor organoids closely resembled primary tumor epithelium. Expression of an epithelial cell marker, E-cadherin, a goblet cell marker, MUC2, and a fibroblast marker, vimentin, but not a myofibroblast marker, α-smooth muscle actin (SMA), was observed in normal organoids. Expression of E-cadherin, MUC2, vimentin, and α-SMA was observed in tumor organoids. Expression of a cancer stem cell marker, LGR5 in tumor organoids, was higher than that in primary tumor tissues. Tumor organoids were more resistant to toxicity of 5-fluorouracil and Irinotecan than colorectal cancer cell lines, SW480, SW620, and HCT116. These findings indicate that ALI organoid culture from colorectal cancer patients may become a novel model that is useful for examining resistance to chemotherapy in tumor microenvironment. PMID:28119740

  18. Functional gait outcomes for idiopathic normal pressure hydrocephalus after primary endoscopic third ventriculostomy.

    PubMed

    Sankey, Eric W; Jusué-Torres, Ignacio; Elder, Benjamin D; Goodwin, C Rory; Batra, Sachin; Hoffberger, Jamie; Lu, Jennifer; Blitz, Ari M; Rigamonti, Daniele

    2015-08-01

    We evaluated if patients with idiopathic normal pressure hydrocephalus (iNPH) showed functional improvement after primary endoscopic third ventriculostomy (ETV). The efficacy of ETV for iNPH remains controversial. We retrospectively reviewed 10 consecutive patients treated between 2009 and 2011 with ETV for iNPH. Seven patients with a median age of 73 years (range: 60-80) who underwent a primary ETV for iNPH were included for analysis. Median follow-up was 39 months (range: 26-46). Post-ETV stoma and aqueductal and cisternal flows were confirmed via high resolution, gradient echo and phase contrast MRI. Post-ETV timed up and go (TUG) and Tinetti performance oriented mobility assessment scores were compared to pre- and post-lumbar puncture (LP) values. A second LP was performed if ETV failed to sustain the observed improvement after initial LP. Patients who demonstrated ETV failure were subsequently shunted. Compared to pre-LP TUG and Tinetti values of 14.00 seconds (range: 12.00-23.00) and 22 (range: 16-24), post-LP scores improved to 11.00 seconds (range: 8.64-15.00; p=0.06) and 25 (range: 24-28; p=0.02), respectively. ETV failed to sustain this improvement with slight worsening between pre-LP and post-ETV TUG and Tinetti scores. Improvement from pre-LP assessment was regained after shunting and at last follow-up with TUG and Tinetti scores of 12.97 seconds (range: 9.00-18.00; p=0.250) and 25 (range: 18-27; p=0.07), and 11.87 seconds (range: 8.27-18.50; p=0.152) and 23 (range: 18-26; p=0.382), respectively. Despite stoma patency, ETV failed to sustain functional improvement seen after LP, however, improvement was regained after subsequent shunting suggesting that shunt placement remains the preferred treatment for iNPH.

  19. Impact of Different Normality Thresholds for 24-hour ABPM at the Primary Health Care Level

    PubMed Central

    Grezzana, Guilherme Brasil; Moraes, David William; Stein, Airton Tetelbon; Pellanda, Lucia Campos

    2017-01-01

    Background Hypertension is an important risk factor for cardiovascular outcomes. Primary health care (PHC) physicians should be prepared to act appropriately in the prevention of cardiovascular risk factors. However, the rates of patients with control of blood pressure (BP) remain low. The impact of the reclassification of high BP by 24-hour ambulatory BP monitoring (ABPM) can lead to different medical decisions in PHC. Objective To evaluate the agreement between the BP measured by a conventional method by PHC physicians and by 24-hour ABPM, considering different BP normal thresholds for the 24-hour ABPM according to the V Brazilian ABPM Guidelines and the European Society of Hypertension Guidelines. Methods A cross-sectional study including 569 hypertensive patients. The BP was initially measured by the PHC physicians and, later, by 24-hour ABPM. The BP measurements were obtained independently between the two methods. The therapeutic targets for the conventional BP followed the guidelines by the Eighth Joint National Committee (JNC 8), the V ABPM Brazilian Guidelines, and the 2013 European Hypertension Guidelines. Results There was an accuracy of 54.8% (95% confidence interval [95%CI] 0.51 - 0.58%) for the BP measured with the conventional method when compared with the 24-hour ABPM, with a sensitivity of 85% (95%CI 80.8 - 88.6%), specificity of 31.9% (95%CI 28.7 - 34.7%), and kappa value of 0.155, when considering the European Hypertension Guidelines. When using more stringent thresholds to characterize the BP as "normal" by ABPM, the accuracy was 45% (95%CI 0.41 - 0.47%) for conventional measurement when compared with 24-hour ABPM, with a sensitivity of 86.7% (95%CI 0.81 - 0.91%), specificity of 29% (95%CI 0.26 - 0.30%), and kappa value of 0.103. Conclusion The BP measurements obtained by PHC physicians showed low accuracy when compared with those obtained by 24-hour ABPM, regardless of the threshold set by the different guidelines. PMID:28099585

  20. Primary outgrowth cultures are a reliable source of human pancreatic stellate cells.

    PubMed

    Han, Song; Delitto, Daniel; Zhang, Dongyu; Sorenson, Heather L; Sarosi, George A; Thomas, Ryan M; Behrns, Kevin E; Wallet, Shannon M; Trevino, Jose G; Hughes, Steven J

    2015-11-01

    Recent advances demonstrate a critical yet poorly understood role for the pancreatic stellate cell (PSC) in the pathogenesis of chronic pancreatitis (CP) and pancreatic cancer (PC). Progress in this area has been hampered by the availability, fidelity, and/or reliability of in vitro models of PSCs. We examined whether outgrowth cultures from human surgical specimens exhibited reproducible phenotypic and functional characteristics of PSCs. PSCs were cultured from surgical specimens of healthy pancreas, CP and PC. Growth dynamics, phenotypic characteristics, soluble mediator secretion profiles and co-culture with PC cells both in vitro and in vivo were assessed. Forty-seven primary cultures were established from 52 attempts, demonstrating universal α-smooth muscle actin and glial fibrillary acidic protein but negligible epithelial surface antigen expression. Modification of culture conditions consistently led to cytoplasmic lipid accumulation, suggesting induction of a quiescent phenotype. Secretion of growth factors, chemokines and cytokines did not significantly differ between donor pathologies, but did evolve over time in culture. Co-culture of PSCs with established PC cell lines resulted in significant changes in levels of multiple secreted mediators. Primary PSCs co-inoculated with PC cells in a xenograft model led to augmented tumor growth and metastasis. Therefore, regardless of donor pathology, outgrowth cultures produce PSCs that demonstrate consistent growth and protein secretion properties. Primary cultures from pancreatic surgical specimens, including malignancies, may represent a reliable source of human PSCs.

  1. Comparison of Prelaminar Thickness between Primary Open Angle Glaucoma and Normal Tension Glaucoma Patients

    PubMed Central

    Jung, Youn Hea; Park, Hae-Young L.; Jung, Kyoung In; Park, Chan Kee

    2015-01-01

    Main Objective The thinning of prelaminar tissue and prelamina cupping is known to occur by ischemia, as we see in anterior ischemic optic neuropathy. Since normal tension glaucoma (NTG) is thought to be more related to vascular factor than in primary open-angle glaucoma (POAG), we hypothesized that prelamina thinning may occur prominently in NTG patients. This study investigated the difference in prelaminar tissue thickness between patients with POAG and NTG and verified the factors related to prelaminar thinning. Methods Complete ophthalmic examination including standard automatic perimetry was performed in all patients. The prelaminar tissue thickness was measured in all patients by performing enhanced depth imaging with a Heidelberg Spectralis Optical Coherence Tomography. The retinal nerve fiber layer and optic nerve head parameters were obtained using the Heidelberg Retina Tomography II and Cirrus Optical Coherence Tomography. Various ocular factors and their relationships with prelaminar thickness were analyzed. Results The mean prelaminar tissue thickness was significantly thinner in patients with POAG than in those with NTG. The difference in the prelaminar thickness between patients with POAG and those with NTG was greater in the early field defect group than in the moderate and severe field groups. In multivariate analysis, the mean prelaminar thickness was related to the intraocular pressure, mean deviation, cup-disc ratio, and cup volume. Conclusions The prelaminar tissue was thinner in patients with POAG than in patients with NTG, and intraocular pressure had a strong influence on the prelaminar thickness in both POAG and NTG. This may indicate that mechanical compression is the main pathogenic factor in both POAG and NTG. PMID:25793734

  2. Ultrastructural and biochemical alterations induced in human, rat and mouse hepatocytes in primary culture exposed to selected carcinogens

    SciTech Connect

    Cole, K.H.

    1987-01-01

    Aflatoxin B1 (AFB{sub 1}), dimethylnitrosamine (DMN), 2-acetylaminofluorene (2-AAF) and actinomycin D are all potential human liver carcinogens. In order to investigate carcinogenic susceptibility of human liver to these agents, primary cultures of normal human hepatocytes were exposed to the four carcinogens. In the first series of experiments, human, rat, and mouse hepatocytes in primary culture were exposed to actinomycin D, AFB{sub 1}, and DMN for 24 h and examined for ultrastructural alterations. In an effort to relate the ultrastructural effects with total covalent binding of the carcinogen to DNA, human, rat and mouse hepatocytes were exposed to 2.0 {times} 10{sup {minus}7} M ({sup 3}H)AFB{sub 1} for 24 h. Hepatocytes from male rats had the highest degree of ({sup 3}H)AFB{sub 1}-DNA binding. Human hepatocytes contained the next highest binding level, while hepatocytes from female rats bound 38 pmoles/mg DNA. The AFB{sub 1}-DNA binding level in mouse hepatocytes was 1.4 pmoles/mg DNA. In similar experiments, human, and male and female rat hepatocytes in primary culture were exposed to the carcinogen 2-acetylamino (9-{sup 14}C)fluorene for 24 h. It was determined that male rat hepatocytes had the highest amount of radiolabeled 2-AAF bound to their DNA, female rats contained 0.57 nmoles/mg DNA, while human hepatocytes contained 0.29 nmoles/mg DNA.

  3. Investigating the establishment of primary cell culture from different abalone (Haliotis midae) tissues

    PubMed Central

    Auzoux-Bordenave, Stéphanie; Niesler, Carola; Roodt-Wilding, Rouvay

    2010-01-01

    The abalone, Haliotis midae, is the most valuable commodity in South African aquaculture. The increasing demand for marine shellfish has stimulated research on the biology and physiology of target species in order to improve knowledge on growth, nutritional requirements and pathogen identification. The slow growth rate and long generation time of abalone restrict efficient design of in vivo experiments. Therefore, in vitro systems present an attractive alternative for short term experimentation. The use of marine invertebrate cell cultures as a standardised and controlled system to study growth, endocrinology and disease contributes to the understanding of the biology of economically important molluscs. This paper investigates the suitability of two different H. midae tissues, larval and haemocyte, for establishing primary cell cultures. Cell cultures are assessed in terms of culture initiation, cell yield, longevity and susceptibility to contamination. Haliotis midae haemocytes are shown to be a more feasible tissue for primary cell culture as it could be maintained without contamination more readily than larval cell cultures. The usefulness of short term primary haemocyte cultures is demonstrated here with a growth factor trial. Haemocyte cultures can furthermore be used to relate phenotypic changes at the cellular level to changes in gene expression at the molecular level. PMID:20680682

  4. Reasoning Abilities in Primary School: A Pilot Study on Poor Achievers vs. Normal Achievers in Computer Game Tasks

    ERIC Educational Resources Information Center

    Dagnino, Francesca Maria; Ballauri, Margherita; Benigno, Vincenza; Caponetto, Ilaria; Pesenti, Elia

    2013-01-01

    This paper presents the results of preliminary research on the assessment of reasoning abilities in primary school poor achievers vs. normal achievers using computer game tasks. Subjects were evaluated by means of cognitive assessment on logical abilities and academic skills. The aim of this study is to better understand the relationship between…

  5. Primary targets in photochemical inactivation of cells in culture

    NASA Astrophysics Data System (ADS)

    Berg, Kristian; Jones, Stuart G.; Prydz, Kristian; Moan, Johan

    1995-01-01

    The mechanisms of photoinactivation of NHIK 3025 cells in culture sensitized by tetrasulfonated phenylporphines (TPPS4) are described). Ultracentrifugation studies on postnuclear supernatants indicated that the intracellular distribution of TPPS4 resembles that of (beta) -N-acetyl-D-glucosaminidase ((beta) -AGA), a lysosomal marker enzyme, and that the cytosolic content of TPPS4 is below the detection limit of the ultracentrifugation method. Upon light exposure more than 90% of TPPS4 was lost from the lysosomal fractions, due to lysosomal rupture. The content of TPPS4 in the postnuclear supernatants was reduced by 30 - 40% upon exposure to light. This is most likely due to binding of TPPS4 to the nuclei, which were removed from the cell extracts before ultracentrifugation, after photochemical treatment. The unpolymerized form of tubulin seems to be an important target for the photochemical inactivation of NHIK 3025 cells. Since TPPS4 is mainly localized in lysosomes it was assumed that a dose of light disrupting a substantial number of lysosomes followed by microtubule depolymerization by nocodazole would enhance the sensitivity of the cells to photoinactivation. This was confirmed by using a colony-forming assay. The increased phototoxic effect exerted by such a treatment regime could be explained by an enhanced sensitivity of tubulin to light. Another cytosolic constituent, lactate dehydrogenase, was not photoinactivated by TPPS4 and light.

  6. Evaluation of PFOS-mediated neurotoxicity in rat primary neurons and astrocytes cultured separately or in co-culture.

    PubMed

    Li, Zhenwei; Liu, Qi; Liu, Chang; Li, Chunna; Li, Yachen; Li, Shuangyue; Liu, Xiaohui; Shao, Jing

    2017-02-01

    Perfluorooctane sulfonate (PFOS) is a potential neurotoxicant reported by epidemiological investigations and experimental studies, while the underlying mechanisms are still unclear. Astrocytes not only support for the construction of neurons, but also conduct neuronal functions through glutamate-glutamine cycle in astrocyte-neuron crosstalk. In the present study, the effect of PFOS exposure on rat primary hippocampal neurons or cortex astrocytes was evaluated. Then the role of the astrocytes in PFOS-induced toxic effect on neurons was explored with astrocyte-neuron co-culture system. Exposure of rat primary hippocampal neurons to PFOS has led to oxidation-antioxidation imbalance, increased apoptosis and abnormal autophagy. The adverse effect of PFOS on rat primary cortex astrocytes manifested in the form of altered extracellular glutamate and glutamine concentrations, decreased glutamine synthase activity, as well as decreased gene expression of glutamine synthase, glutamate transporters and glutamine transporters in the glutamate-glutamine cycle. Especially, the alleviation of PFOS-inhibited neurite outgrowth in neurons could be observed in astrocyte-neuron co-culture system, though the ability of astrocytes in fostering neurite outgrowth was affected by PFOS. These results indicated that both astrocytes and neurons might be the targets of PFOS-induced neurotoxicity, and astrocytes could protect against PFOS-inhibited neurite outgrowth in primary cultured neurons. Our research might render some information in explaining the mechanisms of PFOS-induced neurotoxicity.

  7. Basolateral Cl channels in primary airway epithelial cultures.

    PubMed

    Fischer, Horst; Illek, Beate; Finkbeiner, Walter E; Widdicombe, Jonathan H

    2007-06-01

    Salt and water absorption and secretion across the airway epithelium are important for maintaining the thin film of liquid lining the surface of the airway epithelium. Movement of Cl across the apical membrane involves the CFTR Cl channel; however, conductive pathways for Cl movement across the basolateral membrane have been little studied. Here, we determined the regulation and single-channel properties of the Cl conductance (G(Cl)) in airway surface epithelia using epithelial cultures from human or bovine trachea and freshly isolated ciliated cells from the human nasal epithelium. In Ussing chamber studies, a swelling-activated basolateral G(Cl) was found, which was further stimulated by forskolin and blocked by N-phenylanthranilic acid (DPC) = sucrose > flufenamic acid = niflumic acid = glibenclamide > CdCl(2) = 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) = DIDS = ZnCl(2) > tamoxifen > 4,4'-dinitro-2,2'-stilbene-disulfonate disodium salt (DNDS). In whole cell patch-clamp experiments, three types of G(Cl) were identified: 1) a voltage-activated, DIDS- (but not Cd-) blockable and osmosensitive G(Cl); 2) an inwardly rectifying, hyperpolarization-activated and Cd-sensitive G(Cl); and 3) a forskolin-activated, linear G(Cl), which was insensitive to Cd and DIDS. In cell-attached patch-clamp recordings, the basolateral pole of isolated ciliated cells expressed three types of Cl channels: 1) an outwardly rectifying, swelling-activated Cl channel; 2) a strongly inwardly rectifying Cl channel; and 3) a forskolin-activated, low-conductance channel. We propose that, depending on the driving force for Cl across the apical membrane, basolateral Cl channels confine Cl(-) secretion or support transcellular Cl(-) absorption.

  8. Patient safety culture in primary care: developing a theoretical framework for practical use

    PubMed Central

    Kirk, Susan; Parker, Dianne; Claridge, Tanya; Esmail, Aneez; Marshall, Martin

    2007-01-01

    Objective Great importance has been attached to a culture of safe practice in healthcare organisations, but it has proved difficult to engage frontline staff with this complex concept. The present study aimed to develop and test a framework for making the concept of safety culture meaningful and accessible to managers and frontline staff, and facilitating discussion of ways to improve team/organisational safety culture. Setting Eight primary care trusts and a sample of their associated general practices in north west England. Methods In phase 1 a comprehensive review of the literature and a postal survey of experts helped identify the key dimensions of safety culture in primary care. Semistructured interviews with 30 clinicians and managers explored the application of these dimensions to an established theory of organisational maturity. In phase 2 the face validity and utility of the framework was assessed in 33 interviews and 14 focus groups. Results Nine dimensions were identified through which safety culture is expressed in primary care organisations. Organisational descriptions were developed for how these dimensions might be characterised at five levels of organisational maturity. The resulting framework conceptualises patient safety culture as multidimensional and dynamic, and seems to have a high level of face validity and utility within primary care. It aids clinicians' and managers' understanding of the concept of safety culture and promotes discussion within teams about their safety culture maturity. Conclusions The framework moves the agenda on from rhetoric about the importance of safety culture to a way of understanding why and how the shared values of staff working within a healthcare organisation may be operationalised to create a safe environment for patient care. PMID:17693682

  9. Hydroxylation, conjugation and sulfation of bile acids in primary monolayer cultures of rat hepatocytes

    SciTech Connect

    Princen, H.M.; Meijer, P.

    1988-08-15

    Hydroxylation of lithocholic, chenodeoxycholic, deoxycholic and cholic acids was studied in monolayers of rat hepatocytes cultured for 76 h. The majority of added lithocholic and chenodeoxycholic acids was metabolized to beta-muricholic acid (56-76%). A small part of these bile acids (9%), however, and a considerable amount of deoxycholic and cholic acids (21%) were converted into metabolites more polar than cholic acid in the first culture period. Formation of these compounds decreased during the last day of culture. Bile acids synthesized after addition of (4-/sup 14/C)-cholesterol were almost entirely (97%) sulfated and/or conjugated, predominantly with taurine (54-66%), during culture. Sulfated bile acids were mainly composed of free bile acids. The ability of hepatocytes to sulfurylate bile acids declined with culture age. Thus, rat hepatocytes in primary monolayer culture are capable to sulfurylate bile acids and to hydroxylate trihydroxylated bile acids, suggesting formation of polyhydroxylated metabolites.

  10. Organization Complexity and Primary Care Providers' Perceptions of Quality Improvement Culture Within the Veterans Health Administration.

    PubMed

    Korom-Djakovic, Danijela; Canamucio, Anne; Lempa, Michele; Yano, Elizabeth M; Long, Judith A

    2016-01-01

    This study examined how aspects of quality improvement (QI) culture changed during the introduction of the Veterans Health Administration (VHA) patient-centered medical home initiative and how they were influenced by existing organizational factors, including VHA facility complexity and practice location. A voluntary survey, measuring primary care providers' (PCPs') perspectives on QI culture at their primary care clinics, was administered in 2010 and 2012. Participants were 320 PCPs from hospital- and community-based primary care practices in Pennsylvania, West Virginia, Delaware, New Jersey, New York, and Ohio. PCPs in community-based outpatient clinics reported an improvement in established processes for QI, and communication and cooperation from 2010 to 2012. However, their peers in hospital-based clinics did not report any significant improvements in QI culture. In both years, compared with high-complexity facilities, medium- and low-complexity facilities had better scores on the scales assessing established processes for QI, and communication and cooperation.

  11. Response to hypogravity of normal in vitro cultured follicular cells from thyroid

    NASA Astrophysics Data System (ADS)

    Meli, Antonella; Perrella, Giuseppina; Curcio, Francesco; Saverio, F.; Impiombato, Ambesi

    Aim of this investigation is the study of molecular modifications occurring in differentiated mammalian cells exposed to gravitational changes. The test system chosen is a well characterized clone of differentiated, normal thyroid follicular cells (FRTL5) in long-term culture. As a follow-up to our recent experiment performed during the MASER-7 sounding rocket mission, flown for European Space Agency by Swedish Space Corporation in May 1996, we evaluated FRTL5 cells responses to Thyroid Stimulating Hormone dependent cAMP production under acute hypogravity conditions obtained in a fast rotating clinostat. Following this approach, we evaluated the FRTL5 cells response to TSH under microgravity conditions in order to optimize experimental tools and strategies in preparation to, and in between real flight missions.

  12. Novel approach of signal normalization for depth profile of cultural heritage materials

    NASA Astrophysics Data System (ADS)

    Syvilay, D.; Detalle, V.; Wilkie-Chancellier, N.; Texier, A.; Martinez, L.; Serfaty, S.

    2017-01-01

    The investigation of cultural heritage materials is always complex and specific because unique. Materials are most often heterogeneous and organized in several layers such as mural paintings or corrosion products. The characterization of a complete artwork's stratigraphy is actually one of the questions of science conservation. Indeed, the knowledge of these layers allows completing the history of the work of art and a better understanding of alteration processes in order to set up an appropriate conservation action. The LIBS technique has been employed to study the stratigraphy of an artwork thanks to the ablation laser. However, as we know, atomic information could be insufficient to characterize two materials composed by the same based elements. Therefore, an additional molecular analysis, like Raman spectroscopy; is sometimes necessary for a better identification of the material in particular for organic coatings in cultural heritage. We suggest in this study to use Standard Normal Variate (SNV) as a common normalization for different kinds of spectra (LIBS and Raman spectroscopy) combined with a 3D colour representation for stratigraphic identification of the different layers composing the complex material from artwork. So in this investigation, the SNV method will be applied on LIBS and Raman spectra but also on baseline Raman spectra often considering as nuisance. The aim of this study is to demonstrate the versatility of SNV applied on varied spectra like LIBS, Raman spectra as well as the luminescence background. This original work considers the SNV with a 3D colour representation as a probable new perspective for an easy recognition of a structure layered with a direct overview of the depth profile of the artwork.

  13. Cellular microenvironment dictates androgen production by murine fetal Leydig cells in primary culture.

    PubMed

    Carney, Colleen M; Muszynski, Jessica L; Strotman, Lindsay N; Lewis, Samantha R; O'Connell, Rachel L; Beebe, David J; Theberge, Ashleigh B; Jorgensen, Joan S

    2014-10-01

    Despite the fact that fetal Leydig cells are recognized as the primary source of androgens in male embryos, the mechanisms by which steroidogenesis occurs within the developing testis remain unclear. A genetic approach was used to visualize and isolate fetal Leydig cells from remaining cells within developing mouse testes. Cyp11a1-Cre mice were bred to mT/mG dual reporter mice to target membrane-tagged enhanced green fluorescent protein (GFP) within steroidogenic cells, whereas other cells expressed membrane-tagged tandem-dimer tomato red. Fetal Leydig cell identity was validated using double-labeled immunohistochemistry against GFP and the steroidogenic enzyme 3beta-HSD, and cells were successfully isolated as indicated by qPCR results from sorted cell populations. Because fetal Leydig cells must collaborate with neighboring cells to synthesize testosterone, we hypothesized that the fetal Leydig cell microenvironment defined their capacity for androgen production. Microfluidic culture devices were used to measure androstenedione and testosterone production of fetal Leydig cells that were cultured in cell-cell contact within a mixed population, were isolated but remained in medium contact via compartmentalized co-culture with other testicular cells, or were isolated and cultured alone. Results showed that fetal Leydig cells maintained their identity and steroidogenic activity for 3-5 days in primary culture. Microenvironment dictated proficiency of testosterone production. As expected, fetal Leydig cells produced androstenedione but not testosterone when cultured in isolation. More testosterone accumulated in medium from mixed cultures than from compartmentalized co-cultures initially; however, co-cultures maintained testosterone synthesis for a longer time. These data suggest that a combination of cell-cell contact and soluble factors constitute the ideal microenvironment for fetal Leydig cell activity in primary culture.

  14. Cellular Microenvironment Dictates Androgen Production by Murine Fetal Leydig Cells in Primary Culture1

    PubMed Central

    Carney, Colleen M.; Muszynski, Jessica L.; Strotman, Lindsay N.; Lewis, Samantha R.; O'Connell, Rachel L.; Beebe, David J.; Theberge, Ashleigh B.; Jorgensen, Joan S.

    2014-01-01

    ABSTRACT Despite the fact that fetal Leydig cells are recognized as the primary source of androgens in male embryos, the mechanisms by which steroidogenesis occurs within the developing testis remain unclear. A genetic approach was used to visualize and isolate fetal Leydig cells from remaining cells within developing mouse testes. Cyp11a1-Cre mice were bred to mT/mG dual reporter mice to target membrane-tagged enhanced green fluorescent protein (GFP) within steroidogenic cells, whereas other cells expressed membrane-tagged tandem-dimer tomato red. Fetal Leydig cell identity was validated using double-labeled immunohistochemistry against GFP and the steroidogenic enzyme 3beta-HSD, and cells were successfully isolated as indicated by qPCR results from sorted cell populations. Because fetal Leydig cells must collaborate with neighboring cells to synthesize testosterone, we hypothesized that the fetal Leydig cell microenvironment defined their capacity for androgen production. Microfluidic culture devices were used to measure androstenedione and testosterone production of fetal Leydig cells that were cultured in cell-cell contact within a mixed population, were isolated but remained in medium contact via compartmentalized co-culture with other testicular cells, or were isolated and cultured alone. Results showed that fetal Leydig cells maintained their identity and steroidogenic activity for 3–5 days in primary culture. Microenvironment dictated proficiency of testosterone production. As expected, fetal Leydig cells produced androstenedione but not testosterone when cultured in isolation. More testosterone accumulated in medium from mixed cultures than from compartmentalized co-cultures initially; however, co-cultures maintained testosterone synthesis for a longer time. These data suggest that a combination of cell-cell contact and soluble factors constitute the ideal microenvironment for fetal Leydig cell activity in primary culture. PMID:25143354

  15. Cocaine Causes Apoptotic Death in Rat Mesencephalon and Striatum Primary Cultures.

    PubMed

    Lepsch, Lucilia B; Planeta, Cleopatra S; Scavone, Critoforo

    2015-01-01

    To study cocaine's toxic effects in vitro, we have used primary mesencephalic and striatal cultures from rat embryonic brain. Treatment with cocaine causes a dramatic increase in DNA fragmentation in both primary cultures. The toxicity induced by cocaine was paralleled with a concomitant decrease in the microtubule associated protein 2 (MAP2) and/or neuronal nucleus protein (NeuN) staining. We also observed in both cultures that the cell death caused by cocaine was induced by an apoptotic mechanism, confirmed by TUNEL assay. Therefore, the present paper shows that cocaine causes apoptotic cell death and inhibition of the neurite prolongation in striatal and mesencephalic cell culture. These data suggest that if similar neuronal damage could be produced in the developing human brain, it could account for the qualitative or quantitative defects in neuronal pathways that cause a major handicap in brain function following prenatal exposure to cocaine.

  16. Primary culture of strial marginal cells of guinea pig cochlea: growth, morphologic features, and characterization.

    PubMed

    Achouche, J; Liu, D S; Tran Ba Huy, P; Huy, P T

    1991-12-01

    To further investigate the cellular mechanisms involved in the formation of endolymph, primary cultures of marginal cells of guinea pig were established. Minute explants obtained by mechanical dissociation of stria vascularis were plated on collagen type I precoated impermeable substrate in serum-free, hormone-supplemented medium. A confluent layer of epithelial-like cells was obtained within 2 weeks. The cultured cells formed domes, demonstrating that they retain some of their transepithelial properties. Polarization was also suggested by electron microscopic observation of apical microvilli and tight junctions. Immunohistochemical methods revealed that the cultured cells coexpressed cytokeratin and vimentin, demonstrating their epithelial origin, although some degree of dedifferentiation occurred. Thus, a primary culture of marginal cells can be established that may be a suitable model for an in-depth investigation of the function of the marginal cells.

  17. Cocaine Causes Apoptotic Death in Rat Mesencephalon and Striatum Primary Cultures

    PubMed Central

    Lepsch, Lucilia B.; Planeta, Cleopatra S.; Scavone, Critoforo

    2015-01-01

    To study cocaine's toxic effects in vitro, we have used primary mesencephalic and striatal cultures from rat embryonic brain. Treatment with cocaine causes a dramatic increase in DNA fragmentation in both primary cultures. The toxicity induced by cocaine was paralleled with a concomitant decrease in the microtubule associated protein 2 (MAP2) and/or neuronal nucleus protein (NeuN) staining. We also observed in both cultures that the cell death caused by cocaine was induced by an apoptotic mechanism, confirmed by TUNEL assay. Therefore, the present paper shows that cocaine causes apoptotic cell death and inhibition of the neurite prolongation in striatal and mesencephalic cell culture. These data suggest that if similar neuronal damage could be produced in the developing human brain, it could account for the qualitative or quantitative defects in neuronal pathways that cause a major handicap in brain function following prenatal exposure to cocaine. PMID:26295051

  18. Health system factors affecting communication with pediatricians: gendered work culture in primary care.

    PubMed

    Lynch, Sean

    2011-01-01

    This qualitative study examined the roles that practice setting, education level, and gender may play in social workers' communication satisfaction with pediatricians. Taking an ethnographic approach, the researcher interviewed social workers and pediatricians who worked together to provide mental health services in primary care. The results suggested that gender at the health system level may be an issue and that gendered work culture in primary care was a factor in communication. In particular, reimbursement, an aspect of the gendered work culture, was a substantial communication barrier, and the implications for Medicaid billing are discussed.

  19. The proliferation and differentiation of primary pig preadipocytes is suppressed when cultures are incubated at 37°Celsius compared to euthermic conditions in pigs.

    PubMed

    Bohan, Amy E; Purvis, Katelyn N; Bartosh, Julia L; Brandebourg, Terry D

    2014-01-01

    Given similarities in metabolic parameters and cardiovascular physiology, the pig is well positioned as a biomedical model for metabolic disease and obesity in humans. Better understanding molecular mechanisms governing porcine adipocyte hyperplasia may provide insight into the regulation of adipose tissue development that is useful both when considering the pig as a commodity and when extrapolating porcine data to human disease. Primary cultures of pig stromal-vascular cells have served as a useful tool for investigating factors that regulate preadipocyte proliferation and differentiation. However, such cultures have generally been maintained at 37°C in vitro despite euthermia being 39°C in pigs. To address potential concerns about the physiological relevance of culturing primary pig preadipocytes under what would be hypothermic conditions in vivo, the objective of this study was to investigate the effect of culture temperature on the proliferation and differentiation of pig preadipocytes in primary culture. Culturing primary preadipocytes at 37 rather than 39°C decreases their proliferation rates based upon cleavage of the tetrazolium salt, MTT (P < 0.001), reduction of resazurin (P < 0.001), and daily cell counts (P < 0.001). Likewise, culturing primary porcine preadipocytes at 37°C suppressed their adipogenic potential based upon monitoring adipogenesis morphologically, biochemically, and via the expression of mRNA encoding adipogenic marker genes. Collectively, these data indicate the proliferation and differentiation of primary pig preadipocytes is suppressed when cultures are incubated at 37°C compared to normal body temperature of pigs. This may confound investigation of factors that impact adipocyte hyperplasia in the pig.

  20. Optimization of NRU assay in primary cultures of Eisenia fetida for metal toxicity assessment.

    PubMed

    Irizar, Amaia; Duarte, Daniel; Guilhermino, Lucia; Marigómez, Ionan; Soto, Manu

    2014-09-01

    Coelomocytes, immunocompetent cells of lumbricids, have received special attention for ecotoxicological studies due to their sensibility to pollutants. Their in vitro responses are commonly quantified after in vivo exposure to real or spiked soils. Alternatively, quantifications of in vitro responses after in vitro exposure are being studied. Within this framework, the present study aimed at optimizing the neutral red uptake (NRU) assay in primary culture of Eisenia fetida coelomocytes for its application in soil toxicity testing. Optimized assay conditions were: earthworm depuration for 24 h before retrieving coelomocytes by electric extrusion; 2 × 10(5) seeded cells/well (200 µl) for the NRU assay and incubation for 1 h with neutral red dye. Supplementation of the culture medium with serum was not compatible with the NRU assay, but coelomocytes could be maintained with high viability for 3 days in a serum-free medium without replenishment. Thus, primary cultures were used for 24 h in vitro toxicity testing after exposure to different concentrations of Cd, Cu, Ni and Pb (ranging from 0.1 to 100 μg/ml). Primary cultures were sensitive to metals, the viability declining in a dose-dependent manner. The toxicity rank was, from high to low, Pb > Ni > Cd > Cu. Therefore, it can be concluded that the NRU assay in coelomocytes in primary cultures provides a sensitive and prompt response after in vitro exposure to metals.

  1. Efficient and simple approach to in vitro culture of primary epithelial cancer cells

    PubMed Central

    Janik, Karolina; Popeda, Marta; Peciak, Joanna; Rosiak, Kamila; Smolarz, Maciej; Treda, Cezary; Rieske, Piotr; Stoczynska-Fidelus, Ewelina; Ksiazkiewicz, Magdalena

    2016-01-01

    Primary cancer cells constitute a favourable testing platform for in vitro research in oncology field as they reflect tumour state more accurately than the most commonly employed stable cell lines. Unfortunately, due to limited availability of material and difficulties with protocols validation, primary models are rarely implemented into laboratory practice. We have compared protocols for primary cultures, differing in media components and plate coatings. In terms of culture establishment, application of Geltrex® coating demonstrated equal efficiency to feeder layer (83% compared with 72% successfully established breast and 80% compared with 80% prostate tumour specimens), yet it was substantially less complicated and easier to validate. Both Geltrex® coating and tissue-specific primary cell medium were permanently required to successfully maintain primary epithelial prostate cancer cells (PEPCs) in culture. In case of primary epithelial breast cancer cells (PEBCs), collagen I coating enabled to obtain comparable number of passages to Geltrex® coating (P=0.438). Commercial primary cell media demonstrated lower efficiency than tissue-specific ones (PEPCs–5 compared with 8 and PEBCs–6 compared with 9 passages). Interestingly, both analysed tumour types were unsusceptible to induction of culture lifespan extension when transduced with SV40LT, BMI-1 or hEST2 genes, commonly applied as potential immortalizing agents. In conclusion, the approach based on extracellular matrix reconstitution and tissue-specific primary cell media is easy to validate and provides in vitro expansion sufficient for analytical purposes (approximately 8 passages). Therefore, it may facilitate implementation of hardly available experimental models for a variety of analyses. PMID:27803125

  2. Preventive effect of piracetam and vinpocetine on hypoxia-reoxygenation induced injury in primary hippocampal culture.

    PubMed

    Solanki, P; Prasad, D; Muthuraju, S; Sharma, A K; Singh, S B; Ilavzhagan, G

    2011-04-01

    The present study investigates the potential of Piracetam and Vinpocetine (nootropic drugs, known to possess neuroprotective properties) in preventing hypoxia-reoxygenation induced oxidative stress in primary hippocampal cell culture. The hippocampal culture was exposed to hypoxia (95% N(2), 5% CO(2)) for 3h and followed by 1h of reoxygenation (21% O(2) and 5% CO(2)) at 37 °C. The primary hippocampal cultures were supplemented with the optimum dose of Piracetam and Vinpocetine, independently, and the cultures were divided into six groups, viz. Control/Normoxia, Hypoxia, Hypoxia+Piracetam, Hypoxia+Vinpocetine, Normoxia + Piracetam and Normoxia+Vinpocetine. The cell-viability assays and biochemical oxidative stress parameters were evaluated for each of the six groups. Administration of 1mM Piracetam or 500 nM Vinpocetine significantly prevents the culture from hypoxia-reoxygenation injury when determined by Neutral Red assay, LDH release and Acetylcholine esterase activity. Results showed that Piracetam and Vinpocetine supplementation significantly prevented the fall of mitochondrial membrane potential, rise in ROS generation and reduction in antioxidant levels associated with the hypoxia-reoxygenation injury. In conclusion, the present study establishes that both Piracetam and Vinpocetine give neuroprotection against hypoxia-reoxygenation injury in primary hippocampal cell culture.

  3. Protein-engineered scaffolds for in vitro 3D culture of primary adult intestinal organoids.

    PubMed

    DiMarco, Rebecca L; Dewi, Ruby E; Bernal, Gabriela; Kuo, Calvin; Heilshorn, Sarah C

    2015-10-15

    Though in vitro culture of primary intestinal organoids has gained significant momentum in recent years, little has been done to investigate the impact of microenvironmental cues provided by the encapsulating matrix on the growth and development of these fragile cultures. In this work, the impact of various in vitro culture parameters on primary adult murine organoid formation and growth are analyzed with a focus on matrix properties and geometric culture configuration. The air-liquid interface culture configuration was found to result in enhanced organoid formation relative to a traditional submerged configuration. Additionally, through use of a recombinantly engineered extracellular matrix (eECM), the effects of biochemical and biomechanical cues were independently studied. Decreasing mechanical stiffness and increasing cell adhesivity were found to increase organoid yield. Tuning of eECM properties was used to obtain organoid formation efficiency values identical to those observed in naturally harvested collagen I matrices but within a stiffer construct with improved ease of physical manipulation. Increased ability to remodel the surrounding matrix through mechanical or enzymatic means was also shown to enhance organoid formation. As the engineering and tunability of recombinant matrices is essentially limitless, continued property optimization may result in further improved matrix performance and may help to identify additional microenvironmental cues that directly impact organoid formation, development, differentiation, and functional behavior. Continued culture of primary organoids in recombinant matrices could therefore prove to be largely advantageous in the field of intestinal tissue engineering for applications in regenerative medicine and in vitro tissue mimics.

  4. Control of thyrotropin glycosylation in normal rat pituitary cells in culture: effect of thyrotropin-releasing hormone

    SciTech Connect

    Ponsin, G.; Mornex, R.

    1983-08-01

    Regulation of glycosylation of TSH was studied in primary cultures of normal rat pituitary cells. (3H)Glucosamine or (3H)proline incorporation into immunoprecipitable TSH and trichloroacetic acid-precipitable proteins was measured after incubation periods ranging from 4-72 h. TSH release was assessed by RIA of TSH in the medium. TRH (30 nM) specifically increased the glycosylation of TSH despite the fact that it did not stimulate (3H)proline incorporation into the hormone even after 72 h of continuous labeling. The TRH-stimulated (3H)glucosamine-labeled TSH was completely recovered in the incubation medium. Effective concentrations of TRH were in the same range as those necessary for stimulation of TSH release (10(-10) - 10(-6) M). Somatostatin (50 nM) and T3 (10 microM) antagonized TRH effects on both TSH release and glycosylation. Stages of TSH glycosylation were discriminated by the addition to the culture medium of tunicamycin (10 micrograms/ml) or monensin (25 microM), which are known to inhibit core and terminal glycosylation of proteins, respectively. Medium (3H)glucosamine-labeled TSH was fully glycosylated, whereas a large part of the intracellular hormone was only core glycosylated. This suggests that terminal glycosylation of TSH could be related to hormone secretion. TRH stimulated essentially only terminal glycosylation of TSH. No alteration of core glycosylation of the hormone was observed after TRH treatment. The stimulating effect of TRH on terminal glycosylation of TSH is probably related to its ability to stimulate hormone release.

  5. Spectrum of Cytogenomic Abnormalities Revealed by Array Comparative Genomic Hybridization on Products of Conception Culture Failure and Normal Karyotype Samples.

    PubMed

    Zhou, Qinghua; Wu, Shen-Yin; Amato, Katherine; DiAdamo, Autumn; Li, Peining

    2016-03-20

    Approximately 30% of pregnancies after implantation end up in spontaneous abortions, and 50% of them are caused by chromosomal abnormalities. However, the spectrum of genomic copy number variants (CNVs) in products of conception (POC) and the underlying gene-dosage-sensitive mechanisms causing spontaneous abortions remain largely unknown. In this study, array comparative genomic hybridization (aCGH) analysis was performed as a salvage procedure for 128 POC culture failure (POC-CF) samples and as a supplemental procedure for 106 POC normal karyotype (POC-NK) samples. Chromosomal abnormalities were detected in 10% of POC-CF and pathogenic CNVs were detected in 3.9% of POC-CF and 5.7% of POC-NK samples. Compiled results from this study and relevant case series through a literature review demonstrated an abnormality detection rate (ADR) of 35% for chromosomal abnormalities in POC-CF samples, 3.7% for pathogenic CNVs in POC-CF samples, and 4.6% for pathogenic CNVs in POC-NK samples. Ingenuity Pathway Analysis (IPA) was performed on the genes from pathogenic CNVs found in POC samples. The denoted primary gene networks suggested that apoptosis and cell proliferation pathways are involved in miscarriage. In summary, a similar spectrum of cytogenomic abnormalities was observed in POC culture success and POC-CF samples. A threshold effect correlating the number of dosage-sensitive genes in a chromosome with the observed frequency of autosomal trisomy is proposed. A rationalized approach using firstly fluorescence in situ hybridization (FISH) testing with probes of chromosomes X/Y/18, 13/21, and 15/16/22 for common aneuploidies and polyploidies and secondly aCGH for other cytogenomic abnormalities is recommended for POC-CF samples.

  6. Microbiological and bioinformatics analysis of primary Sjögren’s syndrome patients with normal salivation§

    PubMed Central

    Siddiqui, Huma; Chen, Tsute; Aliko, Ardita; Mydel, Piotr M; Jonsson, Roland; Olsen, Ingar

    2016-01-01

    Background Reduced salivation is considered a major clinical feature of most but not all cases of primary Sjögren’s syndrome (pSS). Reduced saliva flow may lead to changes in the salivary microbiota. These changes have mainly been studied with culture that typically recovers only 65% of the bacteria present. Objective This study was to use high throughput sequencing, covering both cultivated and not-yet-cultivated bacteria, to assess the bacterial microbiota of whole saliva in pSS patients with normal salivation. Methods Bacteria of whole unstimulated saliva from nine pSS patients with normal salivation flow and from nine healthy controls were examined by high throughput sequencing of the hypervariable region V1V2 of 16S rRNA using the 454 GS Junior system. Raw sequence reads were subjected to a species-level, reference-based taxonomy assignment pipeline specially designed for studying the human oral microbial community. Each of the sequence reads was BLASTN-searched against a database consisting of reference sequences representing 1,156 oral and 12,013 non-oral species. Unassigned reads were then screened for high-quality non-chimeras and subjected to de novo species-level operational taxonomy unit (OTU) calling for potential novel species. Downstream analyses, including alpha and beta diversities, were analyzed using the Quantitative Insights into Microbial Ecology (QIIME) pipeline. To reveal significant differences between the microbiota of control saliva and Sjögren’s saliva, a statistical method introduced in Metastats www.metastats.cbcb.umd.edu was used. Results Saliva of pSS patients with normal salivation had a significantly higher frequency of Firmicutes compared with controls (p=0.004). Two other major phyla, Synergistetes and Spirochaetes, were significantly depleted in pSS (p=0.001 for both). In addition, we saw a nearly 17% decrease in the number of genera in pSS (25 vs. 30). While Prevotella was almost equally abundant in both groups (25% in p

  7. Antibiotic Supplements Affect Electrophysiological Properties and Excitability of Rat Hippocampal Pyramidal Neurons in Primary Culture

    PubMed Central

    Bahrami, Farideh; Janahmadi, Mahyar

    2013-01-01

    Introduction: Antibiotic supplements are regularly used in neuronal culture media to control contamination; however, they can interfere with the neuronal excitability and affect electrophysiological properties. Therefore, in this study, the effect of penicillin/streptomycin supplements on the spontaneous electrophysiological activity of hippocampal pyramidal neurons was examined. Methods: Electrophysiological whole-cell patch-clamp recordings from rat hippocampal pyramidal cells in primary culture were performed to investigate the effects of antibiotic supplements on the intrinsic excitability of cultured cells. Results: The present findings indicated that presence of antibiotic supplements (penicillin/streptomycin) in the culture medium altered the intrinsic electrical activity of hippocampal pyramidal neurons in primary culture. These alterations included: 1) depolarized resting membrane potential; 2) a significant enhancement in the after-hyperpolarization amplitude; 3) a significant increase in the area under the action potential and in the decay and rise time of the action potential; 4) a significant broadening of action potential and 5) a significant reduction in the firing frequency. Conclusion: These findings suggest that addition of antibiotic supplements to culture media influences the neuronal excitability and alters the electrophysiological properties of cultured neurons, possibly through changing the ionic conductance underlying neuronal excitability. PMID:23567852

  8. Cabergoline protects dopaminergic neurons against rotenone-induced cell death in primary mesencephalic cell culture.

    PubMed

    Meinel, J; Radad, K; Rausch, W-D; Reichmann, H; Gille, G

    2015-01-01

    In the present study, primary mesencephalic cell cultures prepared from embryonic mouse mesencephala were used to investigate the neuroprotective effect of cabergoline, an ergoline D2 receptor agonist, against the pesticide and neurotoxin rotenone relevant to Parkinson disease (PD). Treatment of cultures with cabergoline alone significantly increased the number of tyrosine hydroxylase immunoreactive (THir) neurons and reduced the release of lactate dehydrogenase (LDH) into the culture medium compared to untreated controls. Against rotenone toxicity, cabergoline significantly rescued degenerating THir neurons, reduced the release of LDH into the culture medium and improved the morphology of surviving THir neurons. The neuroprotective effects afforded by cabergoline were independent of dopaminergic stimulation as blocking of dopamine receptors by the dopamine receptor antagonist sulpiride did not prevent them. Furthermore, rotenone-induced formation of reactive oxygen species (ROS) was significantly reduced by cabergoline. Although cabergoline increased the glutathione (GSH) content in the culture, the protective effect for dopaminergic neurons seemed not to be predominantly mediated by increasing GSH, as depletion of GSH by L-buthionine-(S,R)-sulfoximine (BSO), a GSH biosynthesis inhibitor, did not prevent cabergoline-mediated neuroprotection of THir neurons in rotenone-treated cultures. Moreover, cabergoline significantly increased the ATP/protein ratio in primary mesencephalic cell cultures when added alone or prior to rotenone treatment. These results indicate a neuroprotective effect of cabergoline for dopaminergic neurons against rotenone toxicity. This effect was independent of dopamine receptor stimulation and was at least partially mediated by reducing ROS production and increasing the ATP/protein ratio.

  9. Use of primary cultures of Kenyon cells from bumblebee brains to assess pesticide side effects.

    PubMed

    Wilson, Daniel E; Velarde, Rodrigo A; Fahrbach, Susan E; Mommaerts, Veerle; Smagghe, Guy

    2013-09-01

    Bumblebees are important pollinators in natural and agricultural ecosystems. The latter results in the frequent exposure of bumblebees to pesticides. We report here on a new bioassay that uses primary cultures of neurons derived from adult bumblebee workers to evaluate possible side-effects of the neonicotinoid pesticide imidacloprid. Mushroom bodies (MBs) from the brains of bumblebee workers were dissected and dissociated to produce cultures of Kenyon cells (KCs). Cultured KCs typically extend branched, dendrite-like processes called neurites, with substantial growth evident 24-48 h after culture initiation. Exposure of cultured KCs obtained from newly eclosed adult workers to 2.5 parts per billion (ppb) imidacloprid, an environmentally relevant concentration of pesticide, did not have a detectable effect on neurite outgrowth. By contrast, in cultures prepared from newly eclosed adult bumblebees, inhibitory effects of imidacloprid were evident when the medium contained 25 ppb imidacloprid, and no growth was observed at 2,500 ppb. The KCs of older workers (13-day-old nurses and foragers) appeared to be more sensitive to imidacloprid than newly eclosed adults, as strong effects on KCs obtained from older nurses and foragers were also evident at 2.5 ppb imidacloprid. In conclusion, primary cultures using KCs of bumblebee worker brains offer a tool to assess sublethal effects of neurotoxic pesticides in vitro. Such studies also have the potential to contribute to the understanding of mechanisms of plasticity in the adult bumblebee brain.

  10. Production of avian influenza virus vaccine using primary cell cultures generated from host organs.

    PubMed

    Babar, Mustafeez Mujtaba; Riaz, Muhammad Suleman; Zaidi, Najam-us-Sahar Sadaf; Afzal, Farhan; Farooq, Muhammad Sabir

    2013-06-01

    The global availability of a therapeutically effective influenza virus vaccine during a pandemic remains a major challenge for the biopharmaceutical industry. Long production time, coupled with decreased supply of embryonated chicken eggs (ECE), significantly affects the conventional vaccine production. Transformed cell lines have attained regulatory approvals for vaccine production. Based on the fact that the avian influenza virus would infect the cells derived from its natural host, the viral growth characteristics were studied on chicken embryo-derived primary cell cultures. The viral propagation was determined on avian origin primary cell cultures, transformed mammalian cell lines, and in ECE. A comparison was made between these systems by utilizing various cell culture-based assays. In-vitro substrate susceptibility and viral infection characteristics were evaluated by performing hemagglutination assay (HA), 50 % tissue culture infectious dose (TCID₅₀) and monitoring of cytopathic effects (CPE) caused by the virus. The primary cell culture developed from chicken embryos showed stable growth characteristics with no contamination. HA, TCID₅₀, and CPE exhibited that these cell systems were permissive to viral infection, yielding 2-10 times higher viral titer as compared to mammalian cell lines. Though the viral output from the ECE was equivalent to the chicken cell culture, the time period for achieving it was decreased to half. Some of the prerequisites of inactivated influenza virus vaccine production include generation of higher vial titer, independence from exogenous sources, and decrease in the production time lines. Based on the tests, it can be concluded that chicken embryo primary cell culture addresses these issues and can serve as a potential alternative for influenza virus vaccine production.

  11. Development of Quality Assurance System in Culture and Nation Character Education in Primary Education in Indonesia

    ERIC Educational Resources Information Center

    Susilana, Rudi; Asra

    2013-01-01

    The purpose of national education is to develop skills and build dignified national character and civilization in educating nation life (Act No. 20, 2003). The paper describes a system of quality assurance in culture and character education in primary education. This study employs the six sigma model which consists of the formula DMAIC (Define,…

  12. Resource and Production, A Primary Unit in Cultural Geography. Pupil Text and Workbook and Teacher Manual.

    ERIC Educational Resources Information Center

    Imperatore, William

    This is an instructional unit in cultural geography for the primary grades. The major objective of the unit, which is comprised of a Pupil Text/Workbook and Teacher Manual, is to develop the geographic concepts labeled resource and production. Teaching strategies used include the Pestalozzian method of asking leading questions to draw the students…

  13. Knowing in Primary Physical Education in the UK: Negotiating Movement Culture

    ERIC Educational Resources Information Center

    Ward, Gavin; Quennerstedt, Mikael

    2015-01-01

    This paper aims to understand how pupils and teachers actions-in-context constitute being-a-pupil and being-a-teacher within a primary school physical education (PE) movement culture. Dewey and Bentley's theory of transaction, which views organism-in-environment-as-a-whole, enables the researcher to explore how actions-in-ongoing activities…

  14. Culturally Relevant Literature: What Matters Most to Primary-Age Urban Learners

    ERIC Educational Resources Information Center

    Cartledge, Gwendolyn; Keesey, Susan; Bennett, Jessica G.; Ramnath, Rajiv; Council, Morris R., III.

    2016-01-01

    The ratings and rationales primary-age urban learners gave culturally relevant reading passages was the focus of this descriptive study. First- and second-grade students each read 30 researcher-developed passages reflecting the students' immediate and historical backgrounds. The students rated the passages and gave a reason for their ratings. A…

  15. Promoting Cultural Understandings through Song across the Tasman: Pre-Service Primary Teacher Education

    ERIC Educational Resources Information Center

    Joseph, Dawn; Trinick, Robyn

    2016-01-01

    As tertiary music educators across the Tasman we argue that music, particularly song, is an effective medium for teaching and learning about non-western music when preparing generalist primary Pre-Service Teachers (PSTs). Using "voice" as a portable and accessible vehicle to transmit cultural understandings, we draw on the Zimbabwean…

  16. Primary-Grade Students' Knowledge and Thinking about Shelter as a Cultural Universal.

    ERIC Educational Resources Information Center

    Brophy, Jere; Alleman, Janet

    The traditional K-3 social studies curriculum has focused on food, clothing, shelter, communication, transportation, and other cultural universals. A study was designed to provide information with respect to the topic of shelter, and in the process, to assess claims that primary grade students do not need instruction in the topic because they…

  17. Inhibition of DNA synthesis by chemical carcinogens in cultures of initiated and normal proliferating rat hepatocytes

    SciTech Connect

    Novicki, D.L.; Rosenberg, M.R.; Michalopoulos, G.

    1985-01-01

    Rat hepatocytes in primary culture can be stimulated to replicate under the influence of rat serum and sparse plating conditions. Higher replication rates are induced by serum from two-thirds partially hepatectomized rats. The effects of carcinogens and noncarcinogens on the ability of hepatocytes to synthesize DNA were examined by measuring the incorporation of (3H)thymidine by liquid scintillation counting and autoradiography. Hepatocyte DNA synthesis was not decreased by ethanol or dimethyl sulfoxide at concentrations less than 0.5%. No effect was observed when 0.1 mM ketamine, Nembutal, hypoxanthine, sucrose, ascorbic acid, or benzo(e)pyrene was added to cultures of replicating hepatocytes. Estrogen, testosterone, tryptophan, and vitamin E inhibited DNA synthesis by approximately 50% at 0.1 mM, a concentration at which toxicity was noticeable. Several carcinogens requiring metabolic activation as well as the direct-acting carcinogen N-methyl-N'-nitro-N-nitrosoguanidine interfered with DNA synthesis. Aflatoxin B1 inhibited DNA synthesis by 50% (ID50) at concentrations between 1 X 10(-8) and 1 X 10(-7) M. The ID50 for 2-acetylaminofluorene was between 1 X 10(-7) and 1 X 10(-6) M. Benzo(a)pyrene and 3'-methyl-4-dimethylaminoazobenzene inhibited DNA synthesis 50% between 1 X 10(-5) and 1 X 10(-4) M. Diethylnitrosamine and dimethylnitrosamine (ID50 between 1 X 10(-4) and 5 X 10(-4) M) and 1- and 2-naphthylamine (ID50 between 1 X 10(-5) and 5 X 10(-4) M) caused inhibition of DNA synthesis at concentrations which overlapped with concentrations that caused measurable toxicity.

  18. The cutaneous vasoconstrictor response to venous stasis is normal in subjects with primary Raynaud's disease.

    PubMed

    Edwards, C M; Marshall, J M; Pugh, M

    1999-10-01

    In control subjects and in subjects with primary Raynaud's disease, sudden sound or a mild cool stimulus evokes the pattern of alerting response that includes cutaneous vasoconstriction but vasodilatation in forearm muscle. In control subjects, response habituates on repetition of these stimuli both within experimental sessions and over successive days. However, in subjects with primary Raynaud's disease, the cutaneous vasoconstriction and the muscle vasodilatation persist. We have now tested whether a similar disparity exists for the cutaneous vasoconstriction evoked by venous stasis, a response considered to be a veno-arteriolar reflex mediated by sympathetic fibers, but not requiring transmission through the spinal cord. In 10 subjects with primary Raynaud's disease and in 10 matched controls, a sphygmomanometer cuff on the left arm was inflated to 40 mm Hg for 2 minutes, five times on each of three experimental sessions on days 1, 3, and 5. Cutaneous red cell flux (RCF) was recorded from the pulp and dorsum of the left index finger by using a laser Doppler meter; digital vascular conductance (DCVC) was computed as RCF divided by arterial pressure. The first venous stasis, in session 1, evoked a decrease in pulp and dorsum DCVC in the control and primary Raynaud's subjects. There were no differences between the groups in the magnitudes or durations of these responses. Within session 1, the magnitude of the decrease in DCVC diminished on repetition of venous stasis in the dorsum in controls and in the pulp in primary Raynaud's subjects. We propose these effects reflected the similar reductions in baseline DCVC over time; there was no change in the duration of the responses. Repetition of venous stasis had similar effects in both groups of subjects within sessions 2 and 3. Further, judging from the mean of the responses evoked in each Session the decreases evoked in pulp and dorsum DCVC by venous stasis were fully consistent in magnitude and duration over the

  19. Molecular pathway activation features linked with transition from normal skin to primary and metastatic melanomas in human

    PubMed Central

    Shepelin, Denis; Korzinkin, Mikhail; Vanyushina, Anna; Aliper, Alexander; Borisov, Nicolas; Vasilov, Raif; Zhukov, Nikolay; Sokov, Dmitry; Prassolov, Vladimir; Gaifullin, Nurshat; Zhavoronkov, Alex; Bhullar, Bhupinder; Buzdin, Anton

    2016-01-01

    Melanoma is the most aggressive and dangerous type of skin cancer, but its molecular mechanisms remain largely unclear. For transcriptomic data of 478 primary and metastatic melanoma, nevi and normal skin samples, we performed high-throughput analysis of intracellular molecular networks including 592 signaling and metabolic pathways. We showed that at the molecular pathway level, the formation of nevi largely resembles transition from normal skin to primary melanoma. Using a combination of bioinformatic machine learning algorithms, we identified 44 characteristic signaling and metabolic pathways connected with the formation of nevi, development of primary melanoma, and its metastases. We created a model describing formation and progression of melanoma at the level of molecular pathway activation. We discovered six novel associations between activation of metabolic molecular pathways and progression of melanoma: for allopregnanolone biosynthesis, L-carnitine biosynthesis, zymosterol biosynthesis (inhibited in melanoma), fructose 2, 6-bisphosphate synthesis and dephosphorylation, resolvin D biosynthesis (activated in melanoma), D-myo-inositol hexakisphosphate biosynthesis (activated in primary, inhibited in metastatic melanoma). Finally, we discovered fourteen tightly coordinated functional clusters of molecular pathways. This study helps to decode molecular mechanisms underlying the development of melanoma. PMID:26624979

  20. Molecular pathway activation features linked with transition from normal skin to primary and metastatic melanomas in human.

    PubMed

    Shepelin, Denis; Korzinkin, Mikhail; Vanyushina, Anna; Aliper, Alexander; Borisov, Nicolas; Vasilov, Raif; Zhukov, Nikolay; Sokov, Dmitry; Prassolov, Vladimir; Gaifullin, Nurshat; Zhavoronkov, Alex; Bhullar, Bhupinder; Buzdin, Anton

    2016-01-05

    Melanoma is the most aggressive and dangerous type of skin cancer, but its molecular mechanisms remain largely unclear. For transcriptomic data of 478 primary and metastatic melanoma, nevi and normal skin samples, we performed high-throughput analysis of intracellular molecular networks including 592 signaling and metabolic pathways. We showed that at the molecular pathway level, the formation of nevi largely resembles transition from normal skin to primary melanoma. Using a combination of bioinformatic machine learning algorithms, we identified 44 characteristic signaling and metabolic pathways connected with the formation of nevi, development of primary melanoma, and its metastases. We created a model describing formation and progression of melanoma at the level of molecular pathway activation. We discovered six novel associations between activation of metabolic molecular pathways and progression of melanoma: for allopregnanolone biosynthesis, L-carnitine biosynthesis, zymosterol biosynthesis (inhibited in melanoma), fructose 2, 6-bisphosphate synthesis and dephosphorylation, resolvin D biosynthesis (activated in melanoma), D-myo-inositol hexakisphosphate biosynthesis (activated in primary, inhibited in metastatic melanoma). Finally, we discovered fourteen tightly coordinated functional clusters of molecular pathways. This study helps to decode molecular mechanisms underlying the development of melanoma.

  1. Normal human serum (HS) prevents oxidant-induced lysis of cultured endothelial cells (ECs)

    SciTech Connect

    Callahan, K.S.; Harlan, J.M.

    1986-03-01

    Most studies demonstrating oxidant lysis of cultured ECs are performed in serum-free media or media containing low concentrations of bovine serum. The authors found that HS protects human and bovine ECs from lysis caused by reagent H/sub 2/O/sub 2/ or glucose/glucose oxidase (GO)-generated H/sub 2/O/sub 2/. EC injury was assessed by /sup 51/Cr release, cell detachment, or trypan blue dye exclusion. Protective HS activity was dose-dependent with concentrations greater than or equal to 25% preventing lethal injury. Cytotoxicity at 24 hrs, induced by 20 mU/ml GO, was 90.1 +/- 5.2% without HS vs 1.7 +/- 4.6% with 25% HS present (20 exp). Similar protection was observed with heparinized plasma. Of note, comparable concentrations of bovine serum were devoid of protective activity. Addition of fatty acid-free albumin to the media was also without protective effect. Preliminary characterization showed HS activity was stable to 60/sup 0/C for 30 min, non-dialyzable at 25,000 MW cutoff, and retained in delipidated serum. The HS protection was not merely due to scavenging of exogenous H/sub 2/O/sub 2/ as A23187-induced EC lysis was also prevented by HS. Protective activity was not reproduced by purified cerruloplasmin or transferrin. In conclusion, unidentified factor(s) present in HS protect cultured ECs from oxidant-induced lysis. Since endothelium is normally exposed to 100% plasma, the authors suggest that in vitro studies of oxidant-mediated injury be performed in the presence of HS. Factor(s) in HS may play an important role in modulating oxidant-induced vascular injury in vivo.

  2. Rat Sertoli cells acquire a beta-adrenergic response during primary culture.

    PubMed Central

    Kierszenbaum, A L; Spruill, W A; White, M G; Tres, L L; Perkins, J P

    1985-01-01

    Two-dimensional polyacrylamide gel electrophoresis and the radioligand (-)-[125I]iodopindolol (125I-Pin) have been used to study isoproterenol-dependent protein phosphorylation and beta-adrenergic receptor availability, respectively, in cultured Sertoli cells and freshly isolated seminiferous tubular segments of sexually immature and mature rats. Sertoli cells prepared from sexually immature rats show progressive 125I-Pin binding in primary cultures that correlates with isoproterenol-induced cell shape changes, redistribution of immunoreactive vimentin, and phosphorylation of this intermediate filament protein. The development of 125I-Pin binding to Sertoli cell lysates is blocked by cycloheximide. Seminiferous tubules do not show significant isoproterenol-dependent vimentin phosphorylation nor 125I-Pin binding. However, vimentin phosphorylation can be induced by follicle-stimulating hormone or a cyclic nucleotide analog. This study stresses the need for correlating pharmacological-induced responses observed in Sertoli cell primary cultures with those in the intact seminiferous tubule. Images PMID:2984678

  3. Prolactin mediates neuroprotection against excitotoxicity in primary cell cultures of hippocampal neurons via its receptor.

    PubMed

    Vergara-Castañeda, E; Grattan, D R; Pasantes-Morales, H; Pérez-Domínguez, M; Cabrera-Reyes, E A; Morales, T; Cerbón, M

    2016-04-01

    Recently it has been reported that prolactin (PRL) exerts a neuroprotective effect against excitotoxicity in hippocampus in the rat in vivo models. However, the exact mechanism by which PRL mediates this effect is not completely understood. The aim of our study was to assess whether prolactin exerts neuroprotection against excitotoxicity in an in vitro model using primary cell cultures of hippocampal neurons, and to determine whether this effect is mediated via the prolactin receptor (PRLR). Primary cell cultures of rat hippocampal neurons were used in all experiments, gene expression was evaluated by RT-qPCR, and protein expression was assessed by Western blot analysis and immunocytochemistry. Cell viability was assessed by using the MTT method. The results demonstrated that PRL treatment of neurons from primary cultures did not modify cell viability, but that it exerted a neuroprotective effect, with cells treated with PRL showing a significant increase of viability after glutamate (Glu)--induced excitotoxicity as compared with neurons treated with Glu alone. Cultured neurons expressed mRNA for both PRL and its receptor (PRLR), and both PRL and PRLR expression levels changed after the excitotoxic insult. Interestingly, the PRLR protein was detected as two main isoforms of 100 and 40 kDa as compared with that expressed in hypothalamic cells, which was present only as a 30 kDa variant. On the other hand, PRL was not detected in neuron cultures, either by western blot or by immunohistochemistry. Neuroprotection induced by PRL was significantly blocked by specific oligonucleotides against PRLR, thus suggesting that the PRL role is mediated by its receptor expressed in these neurons. The overall results indicated that PRL induces neuroprotection in neurons from primary cell cultures.

  4. Evidence for multiple bone resorption-stimulating factors produced by normal human keratinocytes in culture.

    PubMed

    Fried, R M; Voelkel, E F; Rice, R H; Levine, L; Tashjian, A H

    1988-06-01

    Conditioned medium from cultured normal human foreskin keratinocytes enhanced the release of calcium from neonatal mouse calvaria in organ culture. Unfractionated keratinocyte-conditioned medium (KCM) stimulated bone resorption in a dose-dependent manner, but it did not increase the concentration of prostaglandin E2 (PGE2) in the bone culture medium until a maximal dose of KCM for resorption was used. Furthermore, inhibitors of PGE2 synthesis, indomethacin, ibuprofen, and piroxicam, did not inhibit KCM-induced calcium release. High concentrations of KCM increased cAMP production by calvaria in the presence of isobutylmethylxanthine, but the increase was small compared with that produced by a dose of bovine PTH that caused a similar level of bone resorption. The bone resorption-stimulating activity of KCM was not lost after incubation at 56 C for 60 min, but it was lost after heating at 100 C for 10 min. Fractionation of KCM by gel filtration chromatography revealed two distinct peaks of bone resorption-stimulating activity. One peak, KCMI, caused a significant increase in bone resorption at 2 micrograms protein/ml. KCMI did not increase medium PGE2, and inhibition of PGE2 synthesis in bone had no effect on KCMI-induced bone resorption. KCMI failed to increase cAMP production by human osteosarcoma SaOS-2 cells. Another peak, KCMII, caused a dose-dependent increase in bone resorption, and a significant increase in medium calcium was noted at a 20-fold lower concentration (0.1 microgram protein/ml) than with KCMI. In contrast to KCMI, the increase in bone resorption stimulated by KCMII was accompanied by a parallel increase in the production of PGE2, and inhibition of PGE2 synthesis completely inhibited the bone resorption-stimulating activity of KCMII. KCMII also caused an increase in cAMP production by SaOS-2 cells. We conclude that KCM contains at least two distinct bone resorption-stimulating factors, one of which acts via a PG-mediated mechanism and the other by

  5. Optic Disc Perfusion in Primary Open Angle and Normal Tension Glaucoma Eyes Using Optical Coherence Tomography-Based Microangiography

    PubMed Central

    Wen, Joanne C.; Zhang, Qinqin; Xin, Chen; Gupta, Divakar; Mudumbai, Raghu C.; Johnstone, Murray A.; Wang, Ruikang K.; Chen, Philip P.

    2016-01-01

    Purpose To investigate optic disc perfusion differences in normal, primary open-angle glaucoma (POAG), and normal tension glaucoma (NTG) eyes using optical microangiography (OMAG) based optical coherence tomography (OCT) angiography technique. Design Cross-sectional, observational study. Subjects Twenty-eight normal, 30 POAG, and 31 NTG subjects. Methods One eye from each subject was scanned with a 68 kHz Cirrus HD-OCT 5,000-based OMAG prototype system centered at the optic nerve head (ONH) (Carl Zeiss Meditec Inc, Dublin, CA). Microvascular images were generated from the OMAG dataset by detecting the differences in OCT signal between consecutive B-scans. The pre-laminar layer (preLC) was isolated by a semi-automatic segmentation program. Main Outcome Measures Optic disc perfusion, quantified as flux, vessel area density, and normalized flux (flux normalized by the vessel area) within the ONH. Results Glaucomatous eyes had significantly lower optic disc perfusion in preLC in all three perfusion metrics (p<0.0001) compared to normal eyes. The visual field (VF) mean deviation (MD) and pattern standard deviation (PSD) were similar between the POAG and NTG groups, and no differences in optic disc perfusion were observed between POAG and NTG. Univariate analysis revealed significant correlation between optic disc perfusion and VF MD, VF PSD, and rim area in both POAG and NTG groups (p≤0.0288). However, normalized optic disc perfusion was correlated with some structural measures (retinal nerve fiber layer thickness and ONH cup/disc ratio) only in POAG eyes. Conclusions Optic disc perfusion detected with OMAG was significantly reduced in POAG and NTG groups compared to normal controls, but no difference was seen between POAG and NTG groups with similar levels of VF damage. Disc perfusion was significantly correlated with VF MD, VF PSD, and rim area in glaucomatous eyes. Vascular changes at the optic disc as measured using OMAG may provide useful information for

  6. Primary cell cultures from sea urchin ovaries: a new experimental tool.

    PubMed

    Mercurio, Silvia; Di Benedetto, Cristiano; Sugni, Michela; Candia Carnevali, M Daniela

    2014-02-01

    In the present work, primary cell cultures from ovaries of the edible sea urchin Paracentrotus lividus were developed in order to provide a simple and versatile experimental tool for researches in echinoderm reproductive biology. Ovary cell phenotypes were identified and characterized by different microscopic techniques. Although cell cultures could be produced from ovaries at all stages of maturation, the cells appeared healthier and viable, displaying a higher survival rate, when ovaries at early stages of gametogenesis were used. In terms of culture medium, ovarian cells were successfully cultured in modified Leibovitz-15 medium, whereas poor results were obtained in minimum essential medium Eagle and medium 199. Different substrates were tested, but ovarian cells completely adhered only on poly-L-lysine. To improve in vitro conditions and stimulate cell proliferation, different serum-supplements were tested. Fetal calf serum and an originally developed pluteus extract were detrimental to cell survival, apparently accelerating processes of cell death. In contrast, cells cultured with sea urchin egg extract appeared larger and healthier, displaying an increased longevity that allowed maintaining them for up to 1 month. Overall, our study provides new experimental bases and procedures for producing successfully long-term primary cell cultures from sea urchin ovaries offering a good potential to study echinoid oogenesis in a controlled system and to investigate different aspects of echinoderm endocrinology and reproductive biology.

  7. Trichostatin A, a critical factor in maintaining the functional differentiation of primary cultured rat hepatocytes

    SciTech Connect

    Henkens, Tom . E-mail: Tom.Henkens@vub.ac.be; Papeleu, Peggy; Elaut, Greetje; Vinken, Mathieu; Rogiers, Vera; Vanhaecke, Tamara

    2007-01-01

    Histone deacetylase inhibitors (HDI) have been shown to increase differentiation-related gene expression in several tumor-derived cell lines by hyperacetylating core histones. Effects of HDI on primary cultured cells, however, have hardly been investigated. In the present study, the ability of trichostatin A (TSA), a prototype hydroxamate HDI, to counteract the loss of liver-specific functions in primary rat hepatocyte cultures has been investigated. Upon exposure to TSA, it was found that the cell viability of the cultured hepatocytes and their albumin secretion as a function of culture time were increased. TSA-treated hepatocytes also better maintained cytochrome P450 (CYP)-mediated phase I biotransformation capacity, whereas the activity of phase II glutathione S-transferases (GST) was not affected. Western blot and qRT-PCR analysis of CYP1A1, CYP2B1 and CYP3A11 protein and mRNA levels, respectively, further revealed that TSA acts at the transcriptional level. In addition, protein expression levels of the liver-enriched transcription factors (LETFs) hepatic nuclear factor 4 alpha (HNF4{alpha}) and CCAAT/enhancer binding protein alpha (C/EBP{alpha}) were accordingly increased by TSA throughout culture time. In conclusion, these findings indicate that TSA plays a major role in the preservation of the differentiated hepatic phenotype in culture. It is suggested that the effects of TSA on CYP gene expression are mediated via controlling the expression of LETFs.

  8. Circadian Rhythms of PER2::LUC in Individual Primary Mouse Hepatocytes and Cultures

    PubMed Central

    Molyneux, Penny C.; Yu, Jimmy K.; Li, Alexander S.; Leise, Tanya L.; Harrington, Mary E.

    2014-01-01

    Background Hepatocytes, the parenchymal cells of the liver, express core clock genes, such as Period2 and Cryptochrome2, which are involved in the transcriptional/translational feedback loop of the circadian clock. Whether or not the liver is capable of sustaining rhythms independent of a central pacemaker is controversial. Whether and how circadian information may be shared among cells in the liver in order to sustain oscillations is currently unknown. Results In this study we isolated primary hepatocytes from transgenic Per2Luc mice and used bioluminescence as a read-out of the state of the circadian clock. Hepatocytes cultured in a collagen gel sandwich configuration exhibited persistent circadian rhythms for several weeks. The amplitude of the rhythms damped, but medium changes consistently reset the phase and amplitude of the cultures. Cry2−/− Per2Luc cells oscillated robustly and expressed a longer period. Co-culturing with wildtype cells did not significantly shorten the period, indicating that coupling among hepatocytes is insufficient to synchronize cells with significantly differing periods. However, spatial patterns revealed by cellular imaging of wildtype cultures provided evidence of weak local coupling among the hepatocytes. Conclusions Our results with primary hepatocyte cultures demonstrate that cultured hepatocytes are weakly coupled. While this coupling is not sufficient to sustain global synchrony, it does increase local synchrony, which may stabilize the circadian rhythms of peripheral oscillators, such as the liver, against noise in the entraining signals. PMID:24498336

  9. Detection of vital germ cell tumor cells in short-term cell cultures of primary tumors and of retroperitoneal metastasis--clinical implications.

    PubMed

    Otto, T; Virchow, S; Fuhrmann, C; Steinberg, F; Streffer, C; Goepel, M; Rübben, H

    1997-01-01

    By establishing short-term cell cultures derived from retroperitoneal metastasis after neoadjuvant chemotherapy, our aim was to improve the diagnosis and prognosis in patients with advanced testicular germ cell tumors. The histological evaluation of surgically removed metastatic tissue by retroperitoneal lymphadenectomy (RLA) is extremely complicated after previous chemotherapy, but knowledge of persistence of vital tumor cells in residual lesions is of great prognostic value and therapeutic consequence in patients with testicular germ cell tumors. We therefore investigated whether vital tumor tissue could be detected in short-term cell cultures derived from such metastatic lesions by measuring the concentration of the tumor markers beta human chorionic gonadotropin (beta HCG) and alpha-1 fetoprotein (AFP) in cell culture supernatants. We initially demonstrated the specificity of the determination in cell cultures of human transitional-cell carcinoma cell lines, human foreskin fibroblasts and normal testicular tissue. In a group of 20 patients with untreated primary testicular germ cell tumors, detection of beta HCG and AFP was increased about threefold in cell culture supernatants in comparison to the serum concentration. Finally, we prepared primary cell cultures from surgically removed retroperitoneal metastasis of 12 patients with testicular germ cell tumors after chemotherapy. The serum concentrations of beta HCG and AFP of all patients were at normal values when RLA was performed. However, pathologically increased concentrations of beta HCG (3/3) and AFP (2/3) in cell culture supernatants were found in 3 of 12 cell cultures. Interestingly, these three patients with a pathological increase in beta HCG and AFP as determined in the supernatant of the short-term cell cultures had tumor progression within a mean follow-up of 3 +/- 1 months (P < 0.01), whereas 9 of 12 patients who had no pathological increase in beta HCG and AFP as determined in the supernatant of

  10. Crouzon syndrome with primary optic nerve atrophy and normal brain functions: A case report

    PubMed Central

    Pal, Uma Shankar; Gupta, Chandan; Chellappa, Arul A.L.

    2012-01-01

    Background This report and review of literature aimed to assess an unusual case of Crouzon syndrome characterized by distinctive disfigurement of craniofacial skeletal and soft tissue structures with primary optic nerve atropy. Methods We present a case of a 12-year-old girl with Crouzon syndrome displaying classic facial abnormalities with reduced vision and hearing loss. Conclusion Crouzon syndrome should be managed as early as possible as it results in airway obstruction, decreased vision, mental retardation and poor cosmetic appearance. PMID:25737846

  11. Thyrotropin Stimulates Differentiation Not Proliferation of Normal Human Thyrocytes in Culture

    PubMed Central

    Morgan, Sarah J.; Neumann, Susanne; Marcus-Samuels, Bernice; Gershengorn, Marvin C.

    2016-01-01

    Although TSH has been suggested to be a proliferative agent for thyrocytes, the effect of TSH on human thyroid cells remains controversial. In particular, most of the reported studies relied primarily on changes in DNA synthesis but have not included measurement of the number of cells. We argue that only a direct count of cell number, demonstrating classical exponential expansion, serves as a valid measurement of proliferation. Thus, although some data support TSH as a proliferative agent, most do not provide conclusive evidence. To generate conclusive evidence with regard to a proliferative effect of TSH in human thyrocytes, we performed various experiments using primary cultures of human thyrocytes. In contrast to previous reports, TSH [±insulin-like growth factor 1 (IGF-1)] did not induce proliferation of thyrocytes under a variety of different conditions. However, TSH/IGF-1 cotreatment did upregulate thyroid-specific gene expression including thyroglobulin (TG) and TSHR in a manner consistent with cellular differentiation. Evidence for a proliferative effect of TSH has been used to inform the American Thyroid Association’s guidelines for the management of thyroid cancer patients, which include TSH suppression. While these recommendations are admittedly based on low- to moderate-quality evidence, TSH suppression is still widely used. We present data that question the consensus view that TSH promotes proliferation of human thyrocytes (upon which the American Thyroid Association’s guidelines are based) and suggest that additional studies, including randomized controlled trials, are warranted to address this important clinical question. PMID:28082948

  12. Picture superiority in free recall: the effects of normal aging and primary degenerative dementia.

    PubMed

    Rissenberg, M; Glanzer, M

    1986-01-01

    A key factor in the decline of memory with age may be a breakdown of communication in the information network involved in memory and cognitive processing. A special case of this communication is assumed to underlie the picture superiority effect in recall. From this hypothesis it follows that the picture superiority effect should lessen with age. In Experiment 1, three groups of adults (young, old normal, and old memory-impaired) were tested in free recall of pictures and word lists. As predicted, the picture superiority effect declined with age. Experiment 2 replicated these findings and showed, moreover, that the picture superiority effect can be reestablished in normal old adults by instructing them to verbalize overtly during item presentation.

  13. Ultraviolet radiation acts as an independent mitogen for normal human melanocytes in culture

    SciTech Connect

    Libow, L.F.; Scheide, S.; DeLeo, V.A.

    1988-01-01

    Identification of growth factors for normal human melanocytes has been significantly aided by the recent development of in vitro culture systems for this cell. Utilizing such a system, we studied the effect of ultraviolet radiation (UVR) on both melanocyte growth and melanization by incorporation of 3H-thymidine and 3H-L-dihydroxyphenylalanine (3H-DOPA), respectively. 3H-thymidine incorporation was found to be significantly stimulated during the first 24 h following a single irradiation. 3H-DOPA incorporation was stimulated after a delay of 2 days postirradiation. Whereas UVR has long been known to induce melanocyte proliferation in vivo, these studies show that UVR can act as a mitogenic stimulus for this cell independent of the cutaneous environment. UVR can thus be added to a growing list of growth factors for epidermal pigment cells and is the only physical agent conclusively shown to act as a mitogen. Included in this list are substances that act via stimulation of the CAMP-kinase or protein kinase systems such as cholera toxin and phorbol esters. UVR is postulated to induce melanocyte proliferation by modulation of these second messenger pathways. With recent evidence linking growth factors, oncogenes and malignant transformation, this study supports the association between UVR exposure and the development of malignant melanoma, and suggests mechanisms whereby UVR may contribute to malignant transformation of this cell.

  14. Primary care units in Emilia-Romagna, Italy: an assessment of organizational culture.

    PubMed

    Pracilio, Valerie P; Keith, Scott W; McAna, John; Rossi, Giuseppina; Brianti, Ettore; Fabi, Massimo; Maio, Vittorio

    2014-01-01

    This study investigates the organizational culture and associated characteristics of the newly established primary care units (PCUs)-collaborative teams of general practitioners (GPs) who provide patients with integrated health care services-in the Emilia-Romagna Region (RER), Italy. A survey instrument covering 6 cultural dimensions was administered to all 301 GPs in 21 PCUs in the Local Health Authority (LHA) of Parma, RER; the response rate was 79.1%. Management style, organizational trust, and collegiality proved to be more important aspects of PCU organizational culture than information sharing, quality, and cohesiveness. Cultural dimension scores were positively associated with certain characteristics of the PCUs including larger PCU size and greater proportion of older GPs. The presence of female GPs in the PCUs had a negative impact on collegiality, organizational trust, and quality. Feedback collected through this assessment will be useful to the RER and LHAs for evaluating and guiding improvements in the PCUs.

  15. Isolation, culture and long-term maintenance of primary mesencephalic dopaminergic neurons from embryonic rodent brains.

    PubMed

    Weinert, Maria; Selvakumar, Tharakeswari; Tierney, Travis S; Alavian, Kambiz N

    2015-02-19

    Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson's diseae. Study of the biological processes involved in physiological functions and vulnerability and death of these neurons is imparative to understanding the underlying causes and unraveling the cure for this common neurodegenerative disorder. Primary cultures of mesDA neurons provide a tool for investigation of the molecular, biochemical and electrophysiological properties, in order to understand the development, long-term survival and degeneration of these neurons during the course of disease. Here we present a detailed method for the isolation, culturing and maintenance of midbrain dopaminergic neurons from E12.5 mouse (or E14.5 rat) embryos. Optimized cell culture conditions in this protocol result in presence of axonal and dendritic projections, synaptic connections and other neuronal morphological properties, which make the cultures suitable for study of the physiological, cell biological and molecular characteristics of this neuronal population.

  16. Multi-cellular 3D human primary liver cell culture elevates metabolic activity under fluidic flow.

    PubMed

    Esch, Mandy B; Prot, Jean-Matthieu; Wang, Ying I; Miller, Paula; Llamas-Vidales, Jose Ricardo; Naughton, Brian A; Applegate, Dawn R; Shuler, Michael L

    2015-05-21

    We have developed a low-cost liver cell culture device that creates fluidic flow over a 3D primary liver cell culture that consists of multiple liver cell types, including hepatocytes and non-parenchymal cells (fibroblasts, stellate cells, and Kupffer cells). We tested the performance of the cell culture under fluidic flow for 14 days, finding that hepatocytes produced albumin and urea at elevated levels compared to static cultures. Hepatocytes also responded with induction of P450 (CYP1A1 and CYP3A4) enzyme activity when challenged with P450 inducers, although we did not find significant differences between static and fluidic cultures. Non-parenchymal cells were similarly responsive, producing interleukin 8 (IL-8) when challenged with 10 μM bacterial lipoprotein (LPS). To create the fluidic flow in an inexpensive manner, we used a rocking platform that tilts the cell culture devices at angles between ±12°, resulting in a periodically changing hydrostatic pressure drop between reservoirs and the accompanying periodically changing fluidic flow (average flow rate of 650 μL min(-1), and a maximum shear stress of 0.64 dyne cm(-2)). The increase in metabolic activity is consistent with the hypothesis that, similar to unidirectional fluidic flow, primary liver cell cultures increase their metabolic activity in response to fluidic flow periodically changes direction. Since fluidic flow that changes direction periodically drastically changes the behavior of other cells types that are shear sensitive, our findings support the theory that the increase in hepatic metabolic activity associated with fluidic flow is either activated by mechanisms other than shear sensing (for example increased opportunities for gas and metabolite exchange), or that it follows a shear sensing mechanism that does not depend on the direction of shear. Our mode of device operation allows us to evaluate drugs under fluidic cell culture conditions and at low device manufacturing and operation

  17. A Simplified Method for Ultra-Low Density, Long-Term Primary Hippocampal Neuron Culture.

    PubMed

    Lu, Zhongming; Piechowicz, Mariel; Qiu, Shenfeng

    2016-03-05

    Culturing primary hippocampal neurons in vitro facilitates mechanistic interrogation of many aspects of neuronal development. Dissociated embryonic hippocampal neurons can often grow successfully on glass coverslips at high density under serum-free conditions, but low density cultures typically require a supply of trophic factors by co-culturing them with a glia feeder layer, preparation of which can be time-consuming and laborious. In addition, the presence of glia may confound interpretation of results and preclude studies on neuron-specific mechanisms. Here, a simplified method is presented for ultra-low density (~2,000 neurons/cm2), long-term (>3 months) primary hippocampal neuron culture that is under serum free conditions and without glia cell support. Low density neurons are grown on poly-D-lysine coated coverslips, and flipped on high density neurons grown in a 24-well plate. Instead of using paraffin dots to create a space between the two neuronal layers, the experimenters can simply etch the plastic bottom of the well, on which the high density neurons reside, to create a microspace conducive to low density neuron growth. The co-culture can be easily maintained for >3 months without significant loss of low density neurons, thus facilitating the morphological and physiological study of these neurons. To illustrate this successful culture condition, data are provided to show profuse synapse formation in low density cells after prolonged culture. This co-culture system also facilitates the survival of sparse individual neurons grown in islands of poly-D-lysine substrates and thus the formation of autaptic connections.

  18. [Nutritional analysis of dietary patterns in students of primary education with normal nutritional status].

    PubMed

    Durá-Gúrpide, Beatriz; Durá-Travé, Teodoro

    2014-06-01

    Objetivo: Realizar un análisis nutricional del modelo dietético en un grupo de alumnos de Educación Primaria (9-12 años) con estado nutricional normal. Material y Métodos: Registro de consumo de alimentos de dos días lectivos consecutivos en una muestra de 353 alumnos de Educación Primaria (188 varones y 165 mujeres) con una situación nutricional normal. Se ha calculado el consumo calórico y de macronutrientes, minerales y vitaminas comparándose con las ingestas recomendadas. Resultados: El valor medio del aporte calórico diario era de 2.066,9 kcal. Los cereales (33%), lácteos (19%) y carnes (17%) aportaban el 70% de la ingesta calórica total. Las proteínas aportaban el 20,3% de la ingesta calórica, los glúcidos el 48,8%, los lípidos el 30,9%, y las grasas saturadas el 12,6%. La ingesta de colesterol era excesiva y 2/3 de la ingesta proteica eran de origen animal. El valor medio de la ingesta de calcio, yodo y vitaminas A, D y E eran inferiores a los aportes dietéticos recomendados. Conclusiones: El modelo dietético de los alumnos de Educación Primaria con estado nutricional normal difiere del prototipo mediterráneo, con un consumo excesivo de carnes, limitado de cereales y lácteos, y deficiente en verduras y hortalizas, frutas, legumbres y pescados; dando lugar a un incremento del aporte de proteínas y grasas animales en detrimento de los hidratos de carbono complejos y un aporte deficiente de calcio, yodo y vitaminas A, D y E.

  19. Basement Membrane Mimics of Biofunctionalized Nanofibers for a Bipolar-Cultured Human Primary Alveolar-Capillary Barrier Model.

    PubMed

    Nishiguchi, Akihiro; Singh, Smriti; Wessling, Matthias; Kirkpatrick, Charles J; Möller, Martin

    2017-03-13

    In vitro reconstruction of an alveolar barrier for modeling normal lung functions and pathological events serve as reproducible, high-throughput pharmaceutical platforms for drug discovery, diagnosis, and regenerative medicine. Despite much effort, the reconstruction of organ-level alveolar barrier functions has failed due to the lack of structural similarity to the natural basement membrane, functionalization with specific ligands for alveolar cell function, the use of primary cells and biodegradability. Here we report a bipolar cultured alveolar-capillary barrier model of human primary cells supported by a basement membrane mimics of fully synthetic bifunctional nanofibers. One-step electrospinning process using a bioresorbable polyester and multifunctional star-shaped polyethylene glycols (sPEG) enables the fabrication of an ultrathin nanofiber mesh with interconnected pores. The nanofiber mesh possessed mechanical stability against cyclic expansion as seen in the lung in vivo. The sPEGs as an additive provide biofunctionality to fibers through the conjugation of peptide to the nanofibers and hydrophilization to prevent unspecific protein adsorption. Biofunctionalized nanofiber meshes facilitated bipolar cultivation of endothelial and epithelial cells with fundamental alveolar functionality and showed higher permeability for molecules compared to microporous films. This nanofiber mesh for a bipolar cultured barrier have the potential to promote growth of an organ-level barrier model for modeling pathological conditions and evaluating drug efficacy, environmental pollutants, and nanotoxicology.

  20. Hyperproliferation of normally quiescent keratinocytes in non-lesional psoriatic skin due to high calcium concentration (an organotypic culture model).

    PubMed

    Szabó, Anna Kenderessy; Bos, J D; Das, P K

    2002-01-01

    Calcium plays an important role in the regulation of different functions of keratinocytes. In the present work we studied the effect of different extracellular calcium concentrations (0.01 mM-2.0 mM) on the proliferation and differentiation of human keratinocytes in normal human and non-lesional psoriatic skin. Using explant culture model, the proliferative and differentiated subsets of keratinocytes were detected by specific antibodies related to cell proliferation [beta-1 integrin (CD29), proliferating cell antigen (Ki67), proliferating cell nuclear antigen (PCNA)] and differentiation [differentiated cell cytokeratins (K1/K10) and differentiating cell antigen (lectin Ulex europaius agglutinin, UEA-1)]. After 4 days of culturing at high Ca2+ (2.0 mM) we observed marked hyperproliferation among the normally quiescent keratinocytes of non-lesional psoriatic skin. In normal uncultured and cultured skin and in uncultured and two-day-cultured non-lesional psoriatic skin both at normal (1.2 mM) and at high (2.0 mM) Ca2+ concentration only one layer of basal CD29+/Ki67+/K1/K10-/UEA-1- cell was observed. In sections from non-lesional psoriatic skin cultured for 4 days in the presence of high Ca2+ (2.0 mM) this cell population has expanded from at least three layers above the basement membrane. This expanded cell population of the 4-day high Ca2+ cultured non-lesional skin showed clear PCNA positive staining on frozen sections with the strongest positivity among the most basal localized cells. These data suggest that (i) extracellular Ca2+ concentration can influence the proliferation of basal ("stem") keratinocytes, (ii) the proliferative response to high Ca2+ concentration of psoriatic non-lesional basal keratinocytes differs from that of normal basal keratinocytes, (iv) changes in the extracellular Ca2+ milieu might play a role in the induction of the hyperproliferative psoriatic lesion.

  1. Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

    PubMed Central

    Meucci, Olimpia

    2011-01-01

    This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or co-culture model. This system is suitable for a variety of experimental needs requiring either a glass or plastic growth substrate and can also be used for culture of other types of neurons. Rat cortical neurons obtained from the late embryonic stage (E17) are plated on glass coverslips or tissue culture dishes facing a feeder layer of glia grown on dishes or plastic coverslips (known as Thermanox), respectively. The choice between the two configurations depends on the specific experimental technique used, which may require, or not, that neurons are grown on glass (e.g. calcium imaging versus Western blot). The glial feeder layer, an astroglia-enriched secondary culture of mixed glia, is separately prepared from the cortices of newborn rat pups (P2-4) prior to the neuronal dissection. A major advantage of this culture system as compared to a culture of neurons only is the support of neuronal growth, survival, and differentiation provided by trophic factors secreted from the glial feeder layer, which more accurately resembles the brain environment in vivo. Furthermore, the co-culture can be used to study neuronal-glial interactions1. At the same time, glia contamination in the neuronal layer is prevented by different means (low density culture, addition of mitotic inhibitors, lack of serum and use of optimized culture medium) leading to a virtually pure neuronal layer, comparable to other established methods1-3. Neurons can be easily separated from the glial layer at any time during culture and used for different experimental applications ranging from electrophysiology4, cellular and molecular biology5-8, biochemistry5, imaging and microscopy4,6,7,9,10. The primary neurons extend axons and dendrites to form functional synapses11, a process which is not observed in neuronal cell lines, although some cell lines do

  2. Establishment of primary bovine intestinal epithelial cell culture and clone method.

    PubMed

    Zhan, Kang; Lin, Miao; Liu, Ming-Mei; Sui, Yang-Nan; Zhao, Guo-Qi

    2017-01-01

    The aim of this study was to establish bovine intestinal epithelial cell (BIEC) line and provide a novel clone cell method. Although various strategies of bovine cell culture and clone techniques have been reported, these methods remain not established. Here, we culture successfully primary BIECs and establish a novel clone cell method. Our result showed that BIECs could be successfully cultured and passaged about generation 5. These cellular aggregates and clusters were adherent loosely at day 2 of culture. Cell aggregates and clusters start to proliferate after approximately 4 d. The BIECs showed positive reaction against cytokeratin 18, E-cadherin, and characteristics of epithelial-like morphology. In addition, the fatty acid-binding proteins (FABPs), villin, and intestinal peptidase (IP) band were positive in BIECs. Our results suggest that the establishment of culturing and clone BIEC methods will apply to isolate and clone other primary cells. These BIECs could therefore contribute to the study of bovine intestinal nutrient absorption and regulation, immune regulation, and the pathogenesis of the bovine intestinal disease, which will provide intestinal cell model in vitro.

  3. Isolation and primary culture of gill and digestive gland cells from the common mussel Mytilus edulis.

    PubMed

    Faucet, Jérôme; Maurice, Manuelle; Gagnaire, Béatrice; Renault, Tristan; Burgeot, Thierry

    2003-01-01

    As the marine mussel Mytilus edulis is commonly used as a sentinel species, it would be useful to develop a primary culture of the target organs most often in contact with the marine environment. This study reports an improved method for dissociating the digestive gland and gills of M. edulis and considers the effect of mussel storage on cell viability and functionality before culture initiation. Viability and enzymatic activities such as those of esterase and peroxidase were monitored by flow cytometry, a sensitive, objective technique allowing large volumes of cells to be counted within a short time. A primary culture of digestive gland showed more than 75% viability after 72 h. Mussels were maintained in an aquarium containing clean, oxygenated seawater at 12 degrees C for two days before culture initiation, and dissociation was performed mechanically and chemically with Ca-Mg-free saline to obtain digestive gland cells. Application of non-specific esterase activity, using fluorescein diacetate (FDA test) coupled with flow cytometry, characterised the functionality of digestive gland and gill cells in culture.

  4. Rapid Genotyping of Animals Followed by Establishing Primary Cultures of Brain Neurons

    PubMed Central

    Huang, Zhengmin; Harata, N. Charles

    2015-01-01

    High-resolution analysis of the morphology and function of mammalian neurons often requires the genotyping of individual animals followed by the analysis of primary cultures of neurons. We describe a set of procedures for: labeling newborn mice to be genotyped, rapid genotyping, and establishing low-density cultures of brain neurons from these mice. Individual mice are labeled by tattooing, which allows for long-term identification lasting into adulthood. Genotyping by the described protocol is fast and efficient, and allows for automated extraction of nucleic acid with good reliability. This is useful under circumstances where sufficient time for conventional genotyping is not available, e.g., in mice that suffer from neonatal lethality. Primary neuronal cultures are generated at low density, which enables imaging experiments at high spatial resolution. This culture method requires the preparation of glial feeder layers prior to neuronal plating. The protocol is applied in its entirety to a mouse model of the movement disorder DYT1 dystonia (ΔE-torsinA knock-in mice), and neuronal cultures are prepared from the hippocampus, cerebral cortex and striatum of these mice. This protocol can be applied to mice with other genetic mutations, as well as to animals of other species. Furthermore, individual components of the protocol can be used for isolated sub-projects. Thus this protocol will have wide applications, not only in neuroscience but also in other fields of biological and medical sciences. PMID:25742545

  5. Organelle Transport in Cultured Drosophila Cells: S2 Cell Line and Primary Neurons.

    PubMed Central

    Gelfand, Vladimir I.

    2013-01-01

    Drosophila S2 cells plated on a coverslip in the presence of any actin-depolymerizing drug form long unbranched processes filled with uniformly polarized microtubules. Organelles move along these processes by microtubule motors. Easy maintenance, high sensitivity to RNAi-mediated protein knock-down and efficient procedure for creating stable cell lines make Drosophila S2 cells an ideal model system to study cargo transport by live imaging. The results obtained with S2 cells can be further applied to a more physiologically relevant system: axonal transport in primary neurons cultured from dissociated Drosophila embryos. Cultured neurons grow long neurites filled with bundled microtubules, very similar to S2 processes. Like in S2 cells, organelles in cultured neurons can be visualized by either organelle-specific fluorescent dyes or by using fluorescent organelle markers encoded by DNA injected into early embryos or expressed in transgenic flies. Therefore, organelle transport can be easily recorded in neurons cultured on glass coverslips using living imaging. Here we describe procedures for culturing and visualizing cargo transport in Drosophila S2 cells and primary neurons. We believe that these protocols make both systems accessible for labs studying cargo transport. PMID:24300413

  6. Age- and Gender-Normalized Coronary Incidence and Mortality Risks in Primary and Secondary Prevention

    PubMed Central

    Puddu, Paolo Emilio; Iannetta, Loredana; Schiariti, Michele

    2012-01-01

    Epidemiologic differences in ischemic heart disease incidence between women and men remain largely unexplained. The reasons of women’s “protection” against coronary artery disease (CAD) are not still clear. However, there are subsets more likely to die of a first myocardial infarction. The purpose of this review is to underline different treatment strategies between genders and describe the role of classical and novel factors defined to evaluate CAD risk and mortality, aimed at assessing applicability and relevance for primary and secondary prevention. Women and men present different age-related risk patterns: it should be important to understand whether standard factors may index CAD risk, including mortality, in different ways and/or whether specific factors might be targeted gender-wise. Take home messages include: HDL-cholesterol levels, higher in pre-menopausal women than in men, are more strictly related to CAD. The same is true for high triglycerides and Lp(a). HDL-cholesterol levels are inversely related to incidence and mortality. In primary prevention the role of statins is not completely ascertained in women although in secondary prevention these agents are equally effective in both genders. Weight and glycemic control are effective to reduce cardiovascular disease (CVD) mortality in women from middle to older age. Blood pressure is strongly and directly related to CVD mortality, from middle to older age, particularly in diabetic and over weighted women. Kidney dysfunction, defined using UAE and eGFR predicts primary CVD incidence and risk in both genders. In secondary prediction, kidney dysfunction predicts sudden death in women in conjunction with left ventricular ejection fraction evaluation. Serum uric acid does not differentiate gender-related CVD incidences, although it increases with age. Age-related differences between genders have been related to loss of ovarian function traditionally and to lower iron stores more recently. QT interval

  7. Lithium treatment elongates primary cilia in the mouse brain and in cultured cells

    SciTech Connect

    Miyoshi, Ko; Kasahara, Kyosuke; Miyazaki, Ikuko; Asanuma, Masato

    2009-10-30

    The molecular mechanisms underlying the therapeutic effects of lithium, a first-line antimanic mood stabilizer, have not yet been fully elucidated. Treatment of the algae Chlamydomonas reinhardtii with lithium has been shown to induce elongation of their flagella, which are analogous structures to vertebrate cilia. In the mouse brain, adenylyl cyclase 3 (AC3) and certain neuropeptide receptors colocalize to the primary cilium of neuronal cells, suggesting a chemosensory function for the primary cilium in the nervous system. Here we show that lithium treatment elongates primary cilia in the mouse brain and in cultured cells. Brain sections from mice chronically fed with Li{sub 2}CO{sub 3} were subjected to immunofluorescence study. Primary cilia carrying both AC3 and the receptor for melanin-concentrating hormone (MCH) were elongated in the dorsal striatum and nucleus accumbens of lithium-fed mice, as compared to those of control animals. Moreover, lithium-treated NIH3T3 cells and cultured striatal neurons exhibited elongation of the primary cilia. The present results provide initial evidence that a psychotropic agent can affect ciliary length in the central nervous system, and furthermore suggest that lithium exerts its therapeutic effects via the upregulation of cilia-mediated MCH sensing. These findings thus contribute novel insights into the pathophysiology of bipolar mood disorder and other psychiatric diseases.

  8. Nanofabricated Collagen-Inspired Synthetic Elastomers for Primary Rat Hepatocyte Culture

    PubMed Central

    Bettinger, Christopher J.; Kulig, Katherine M.; Vacanti, Joseph P.

    2009-01-01

    Synthetic substrates that mimic the properties of extracellular matrix proteins hold significant promise for use in systems designed for tissue engineering applications. In this report, we designed a synthetic polymeric substrate that is intended to mimic chemical, mechanical, and topological characteristics of collagen. We found that elastomeric poly(ester amide) substrates modified with replica-molded nanotopographic features enhanced initial attachment, spreading, and adhesion of primary rat hepatocytes. Further, hepatocytes cultured on nanotopographic substrates also demonstrated reduced albumin secretion and urea synthesis, which is indicative of strongly adherent hepatocytes. These results suggest that these engineered substrates can function as synthetic collagen analogs for in vitro cell culture. PMID:18847357

  9. Comparative Metabolism, Cytotoxicity, and Genotoxicity of Chemical Carcinogens in Primary Cultures of Hepatocytes

    DTIC Science & Technology

    1985-05-01

    death and regeneration. Cytotoxicity is easily assessed in primary hepatocyte cultures by light microscopic observation (Green ott al., 1981) and/or the...The recent development of a sensitive scintillation detection of repair provides a three day assay (Loury and Byard, 1983). A UV light positive control...RATS FED 0.5% BUTYLATED HYDROXYTOLUENEa Amount of ascron.•,cut./cultur-b P10o0. Al’"I boumd/t ,acrtmooletuloc Ratlo of bound producto k’ontro I WN

  10. Isolated Primary Blast Inhibits Long-Term Potentiation in Organotypic Hippocampal Slice Cultures.

    PubMed

    Vogel, Edward W; Effgen, Gwen B; Patel, Tapan P; Meaney, David F; Bass, Cameron R Dale; Morrison, Barclay

    2016-04-01

    Over the last 13 years, traumatic brain injury (TBI) has affected over 230,000 U.S. service members through the conflicts in Iraq and Afghanistan, mostly as a result of exposure to blast events. Blast-induced TBI (bTBI) is multi-phasic, with the penetrating and inertia-driven phases having been extensively studied. The effects of primary blast injury, caused by the shockwave interacting with the brain, remain unclear. Earlier in vivo studies in mice and rats have reported mixed results for primary blast effects on behavior and memory. Using a previously developed shock tube and in vitro sample receiver, we investigated the effect of isolated primary blast on the electrophysiological function of rat organotypic hippocampal slice cultures (OHSC). We found that pure primary blast exposure inhibited long-term potentiation (LTP), the electrophysiological correlate of memory, with a threshold between 9 and 39 kPa·ms impulse. This deficit occurred well below a previously identified threshold for cell death (184 kPa·ms), supporting our previously published finding that primary blast can cause changes in brain function in the absence of cell death. Other functional measures such as spontaneous activity, network synchronization, stimulus-response curves, and paired-pulse ratios (PPRs) were less affected by primary blast exposure, as compared with LTP. This is the first study to identify a tissue-level tolerance threshold for electrophysiological changes in neuronal function to isolated primary blast.

  11. Normalizing the Fraughtness: How Emotion, Race, and School Context Complicate Cultural Competence

    ERIC Educational Resources Information Center

    Buehler, Jennifer; Ruggles Gere, Anne; Dallavis, Christian; Shaw Haviland, Victoria

    2009-01-01

    Preservice teachers seeking to develop cultural competence can face a struggle fraught with multiple challenges, even when they are committed to culturally relevant pedagogy. This article closely analyzes one White beginning teacher's negotiations with cultural competence during a lesson in her student teaching semester, then traces how she made…

  12. Metallothionein-3 regulates lysosomal function in cultured astrocytes under both normal and oxidative conditions.

    PubMed

    Lee, Sook-Jeong; Park, Mi-Ha; Kim, Hyun-Jae; Koh, Jae-Young

    2010-08-01

    Cellular zinc plays a key role in lysosomal change and cell death in neurons and astrocytes under oxidative stress. Here, using astrocytes lacking metallothionein-3 (MT3), a potential source of labile zinc in the brain, we studied the role of MT3 in oxidative stress responses. H(2)O(2) induced a large increase in labile zinc in wild-type (WT) astrocytes, but stimulated only a modest rise in MT3-null astrocytes. In addition, H(2)O(2)-induced lysosomal membrane permeabilization (LMP) and cell death were comparably attenuated in MT3-null astrocytes. Expression and glycosylation of Lamp1 (lysosome-associated membrane protein 1) and Lamp2 were increased in MT3-null astrocytes, and the activities of several lysosomal enzymes were significantly reduced, indicating an effect of MT3 on lysosomal components. Consistent with lysosomal dysfunction in MT3-null cells, the level of LC3-II (microtubule-associated protein 1 light chain 3), a marker of early autophagy, was increased by oxidative stress in WT astrocytes, but not in MT3-null cells. Similar changes in Lamp1, LC3, and cathepsin-D were induced by the lysosomal inhibitors bafilomycin A1, chloroquine, and monensin, indicating that lysosomal dysfunction may lie upstream of changes observed in MT3-null astrocytes. Consistent with this idea, lysosomal accumulation of cholesterol and lipofuscin were augmented in MT3-null astrocytes. Similar to the results seen in MT3-null cells, MT3 knockdown by siRNA inhibited oxidative stress-induced increases in zinc and LMP. These results indicate that MT3 may play a key role in normal lysosomal function in cultured astrocytes.

  13. Recent Developments in Understanding the Role of Aqueous Humor Outflow in Normal and Primary Open Angle Glaucoma

    PubMed Central

    Hann, Cheryl R.; Fautsch, Michael P.

    2015-01-01

    Primary open angle glaucoma (POAG) is the second leading cause of blindness in the world's rapidly aging population. POAG is characterized by progressive degeneration of neural structures in the posterior segment, often associated with a concomitant elevation of intraocular pressure. Changes in IOP are believed to be caused by a disruption in the normal outflow of aqueous humor. This article reviews recent research associated with normal and POAG aqueous humor outflow. Novel findings elucidating biochemical and pathological changes in the ocular tissues affected in POAG are presented. Stem cell research, identification of lymphatic markers, and increased use of mouse models give researchers exciting new tools to understand aqueous humor outflow, changes associated with POAG and identify underlying causes of the disease. PMID:26236568

  14. Expression profile of the matricellular protein osteopontin in primary open-angle glaucoma and the normal human eye.

    PubMed

    Chowdhury, Uttio Roy; Jea, Seung-Youn; Oh, Dong-Jin; Rhee, Douglas J; Fautsch, Michael P

    2011-08-16

    PURPOSE. To characterize the role of osteopontin (OPN) in primary open-angle glaucoma (POAG) and normal eyes. METHODS. OPN quantification was performed by enzyme-linked immunosorbent assay in aqueous humor (AH) obtained from human donor eyes (POAG and normal) and surgical samples (POAG and elective cataract removal). OPN expression and localization in whole eye tissue sections and primary normal human trabecular meshwork (NTM) cells were studied by Western blot and immunohistochemistry. Latanoprost-free acid (LFA)-treated NTM cells were analyzed for OPN gene and protein expression. Intraocular pressure was measured by tonometry, and central corneal thickness was measured by optical coherence tomography in young OPN(-/-) and wild-type mice. RESULTS. OPN levels were significantly reduced in donor POAG AH compared with normal AH (0.54 ± 0.18 ng/μg [n = 8] vs. 0.77 ± 0.23 ng/μg [n = 9]; P = 0.039). A similar trend was observed in surgical AH (1.05 ± 0.31 ng/μg [n = 20] vs. 1.43 ± 0.88 ng/μg [n = 20]; P = 0.083). OPN was present in the trabecular meshwork, corneal epithelium and endothelium, iris, ciliary body, retina, vitreous humor, and optic nerve. LFA increased OPN gene expression, but minimal change in OPN protein expression was observed. No difference in intraocular pressure (17.5 ± 2.0 mm Hg [n = 56] vs. 17.3 ± 1.9 mm Hg [n = 68]) but thinner central corneal thickness (91.7 ± 3.6 μm [n = 50] vs. 99.2 ± 5.5 μm [n = 70]) was noted between OPN(-/-) and wild-type mice. CONCLUSIONS. OPN is widely distributed in the human eye and was found in lower concentrations in POAG AH. Reduction of OPN in young mice does not affect IOP.

  15. [The features of postsynaptic currents in primary culture of rat cortical neurons].

    PubMed

    Sibarov, D A; Antonov, S M

    2013-06-01

    The generation features of postsynaptic currents were studied in primary culture of cortical neurons at 7-20 days in vitro (DIV). The use of specific blockers of postsynaptic ion channels after 10 DIV revealed all types of electrical activity found in adult cortex including miniature inhibitory (mIPSCs), excitatory (mEPSCs) and spontaneous giant excitatory currents and spikes. The frequency of mEPSCs increased exponentially from 7 to 20 DIV doubling every 2.2 days in parallel with changes in action potentials generation. The mEPSCs generated by NMDA and AMPA or by only AMPA receptor activation were found. The inhibition of NMDA receptors by magnesium ions or AP5 were shown to modulate the frequency and amplitude of mEPSCs, which differ primary culture from brain slices possibly because of the lack of glial control of synaptic transmission.

  16. Raman spectroscopy analysis of differences in composition of spent culture media of in vitro cultured preimplantation embryos isolated from normal and fat mice dams.

    PubMed

    Fabian, Dušan; Kačmarová, Martina; Kubandová, Janka; Čikoš, Štefan; Koppel, Juraj

    2016-06-01

    The aim of the present study was to compare overall patterns of metabolic activity of in vitro cultured preimplantation embryos isolated from normal and fat mice dams by means of non-invasive profiling of spent culture media using Raman spectroscopy. To produce females with two different types of body condition (normal and fat), a previously established two-generation model was used, based on overfeeding of experimental mice during prenatal and early postnatal development. Embryos were isolated from spontaneously ovulating and naturally fertilized dams at the 2-cell stage of development and cultured to the blastocyst stage in synthetic oviductal medium KSOMaa. Embryos from fat mice (displaying significantly elevated body weight and fat) showed similar developmental capabilities in vitro as embryos isolated from normal control dams (displaying physiological body weight and fat). The results show that alterations in the composition of culture medium caused by the presence of developing mouse preimplantation embryos can be detected using Raman spectroscopy. Metabolic activity of embryos was reflected in evident changes in numerous band intensities in the 1620-1690cm(-1) (amide I) region and in the 1020-1140cm(-1) region of the Raman spectrum for KSOMaa. Moreover, multivariate analysis of spectral data proved that the composition of proteins and other organic compounds in spent samples obtained after the culture of embryos isolated from fat dams was different from that in spent samples obtained after the culture of embryos from control dams. This study demonstrates that metabolic activity of cultured preimplantation embryos might depend on the body condition of their donors.

  17. Habitus and Flow in Primary School Musical Practice: Relations between Family Musical Cultural Capital, Optimal Experience and Music Participation

    ERIC Educational Resources Information Center

    Valenzuela, Rafael; Codina, Nuria

    2014-01-01

    Based on Bourdieu's idea that cultural capital is strongly related to family context, we describe the relations between family musical cultural capital and optimal experience during compulsory primary school musical practice. We analyse whether children from families with higher levels of musical cultural capital, and specifically with regard to…

  18. The Cultural Aspect of Foreign Languages Teaching at Primary School in Turkey

    ERIC Educational Resources Information Center

    Sadik, Turkoglu; Omer, Kocer

    2011-01-01

    Learning a foreign language is a new concept at primary school in Turkey. Even if the Minister of National Education has been thinking of it for a long time, it has only been compulsory since 2006. Pedagogues are still doing research on the way to teach a new language to children. One of the ideas is to base this new learning on cultural contents.…

  19. Growth and antrum formation of bovine primary follicles in long-term culture in vitro.

    PubMed

    Sun, Jing; Li, Xiangdong

    2013-09-01

    Successful antral formation in vitro from bovine preantral follicles (145-170 μm) has been described previously, but antrum formation from the primary follicle (50-70 μm) has not yet been achieved in vitro. The aim of the study was to establish an optimal culture system supporting the growth and maturation of bovine primary follicles (50-70 μm) in vitro. Bovine primary follicles were cultured in a three-dimensional culture system for 13 or 21 days in alpha-minimum essential medium. Various treatments including follicle stimulating hormone (FSH), luteinizing hormone (LH), 17β-estradiol (E2), basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) were tested. The follicular diameter and antrum formation rate were recorded, and follicular maturation markers (P450 aromatase, CYP19A1; anti-Mullerian hormone, AMH; growth differentiation factor-9, GDF9; bone morphogenetic protein-15, BMP15; and type III transforming growth factor β receptor, TGFβR3) were analyzed by real-time RT-PCR. After 21 days of culture under each treatment condition, the follicular diameter was significantly enlarged in the presence of FSH + LH + E2 + bFGF or FSH + LH + E2 + bFGF + EGF (p<0.05). An addition of 50 ng/ml bFGF or bFGF +25 ng/ml EGF initiated antrum formation by day 19 and day 17 of culture, and the antral cavity formation rate was 16.7% and 33.3% by 21 days of culture, respectively. The expression of follicular maturation markers (CYP19A1, AMH, GDF9, BMP15 and TGFβR3) was significantly altered. We conclude that addition of 50 ng/ml bFGF +25 ng/ml EGF to media containing FSH + LH + E2 turned out to be the most effective optimized culture conditions to support the growth and maturation of bovine primary follicles in vitro.

  20. Quantification and Characterization of UVB-Induced Mitochondrial Fragmentation in Normal Primary Human Keratinocytes

    PubMed Central

    Jugé, Romain; Breugnot, Josselin; Da Silva, Célia; Bordes, Sylvie; Closs, Brigitte; Aouacheria, Abdel

    2016-01-01

    UV irradiation is a major environmental factor causing skin dryness, aging and cancer. UVB in particular triggers cumulative DNA damage, oxidative stress and mitochondrial dysfunction. The objective of our study was to provide both qualitative and quantitative analysis of how mitochondria respond to UVB irradiation in normal human epidermal keratinocytes (NHEK) of healthy donors, with the rationale that monitoring mitochondrial shape will give an indication of cell population fitness and enable the screening of bioactive agents with UVB-protective properties. Our results show that NHEK undergo dose-dependent mitochondrial fragmentation after exposure to UVB. In order to obtain a quantitative measure of this phenomenon, we implemented a novel tool for automated quantification of mitochondrial morphology in live cells based on confocal microscopy and computational calculations of mitochondrial shape descriptors. This method was used to substantiate the effects on mitochondrial morphology of UVB irradiation and of knocking-down the mitochondrial fission-mediating GTPase Dynamin-related protein 1 (DRP1). Our data further indicate that all the major mitochondrial dynamic proteins are expressed in NHEK but that their level changes were stronger after mitochondrial uncoupler treatment than following UVB irradiation or DRP1 knock-down. Our system and procedures might be of interest for the identification of cosmetic or dermatologic UVB-protective agents. PMID:27731355

  1. Characterisation and metabolism of astroglia-rich primary cultures from cathepsin K-deficient mice.

    PubMed

    Dauth, Stephanie; Schmidt, Maike M; Rehders, Maren; Dietz, Frank; Kelm, Sørge; Dringen, Ralf; Brix, Klaudia

    2012-09-01

    Cathepsin K is important for the brain, because its deficiency in mice is associated with a marked decrease in differentiated astrocytes and changes in neuronal patterning in the hippocampus as well as with learning and memory deficits. As cathepsin K activity is most prominent in hippocampal regions of wild type animals, we hypothesised alterations in astrocyte-mediated support of neurons as a potential mechanism underlying the impaired brain functions in cathepsin K-deficient mice. To address this hypothesis, we have generated and characterised astroglia-rich primary cell cultures from cathepsin K-deficient and wild type mice and compared these cultures for possible changes in metabolic support functions and cell composition. Interestingly, cells expressing the oligodendrocytic markers myelin-associated glycoprotein and myelin basic protein were more frequent in astroglia-rich cultures from cathepsin K-deficient mice. However, cell cultures from both genotypes were morphologically comparable and similar with respect to glucose metabolism. In addition, specific glutathione content, glutathione export and γ-glutamyl-transpeptidase activity remained unchanged, whereas the specific activities of glutathione reductase and glutathione-S-transferase were increased by around 50% in cathepsin K-deficient cultures. Thus, lack of cathepsin K in astroglia-rich cultures appears not to affect metabolic supply functions of astrocytes but to facilitate the maturation of oligodendrocytes.

  2. Are primary healthcare services culturally appropriate for Aboriginal people? Findings from a remote community.

    PubMed

    Smith, Kaye; Fatima, Yaqoot; Knight, Sabina

    2017-04-13

    This study explored the views of key stakeholders on cultural appropriateness of primary health care (PHC) services for Aboriginal people. A total of 78 participants, including healthcare providers, administrative team members (n=24, ~30% of study sample) and Aboriginal community members (n=54, ~70% of study sample) living in remote North West Queensland participated in the study. Outcome measures were assessed by administering survey questionnaires comprising qualitative questions and various subscales (e.g. provider behaviours and attitudes, communication, physical environment and facilities, and support from administrative staff). Descriptive statistics were used to present quantitative findings, whereas inductive thematic analysis was used for qualitative data. In contrast to the views of PHC providers, a significant number of Aboriginal people did not perceive that they were receiving culturally appropriate services. Although PHC providers acknowledged cultural awareness training for familiarising themselves with Aboriginal culture, they found the training to be general, superficial and lacking prospective evaluation. PHC providers should understand that culturally inappropriate clinical encounters generate mistrust and dissatisfaction. Therefore, a broad approach involving culturally respectful association between PHC providers, Aboriginal consumers and administrative staff is required to bring sustainable changes at the practice level to improve the health of Aboriginal people.

  3. Diabetes increases susceptibility of primary cultures of rat proximal tubular cells to chemically induced injury

    SciTech Connect

    Zhong Qing; Terlecky, Stanley R.; Lash, Lawrence H.

    2009-11-15

    Diabetic nephropathy is characterized by increased oxidative stress and mitochondrial dysfunction. In the present study, we prepared primary cultures of proximal tubular (PT) cells from diabetic rats 30 days after an ip injection of streptozotocin and compared their susceptibility to oxidants (tert-butyl hydroperoxide, methyl vinyl ketone) and a mitochondrial toxicant (antimycin A) with that of PT cells isolated from age-matched control rats, to test the hypothesis that PT cells from diabetic rats exhibit more cellular and mitochondrial injury than those from control rats when exposed to these toxicants. PT cells from diabetic rats exhibited higher basal levels of reactive oxygen species (ROS) and higher mitochondrial membrane potential, demonstrating that the PT cells maintain the diabetic phenotype in primary culture. Incubation with either the oxidants or mitochondrial toxicant resulted in greater necrotic and apoptotic cell death, greater evidence of morphological damage, greater increases in ROS, and greater decreases in mitochondrial membrane potential in PT cells from diabetic rats than in those from control rats. Pretreatment with either the antioxidant N-acetyl-L-cysteine or a catalase mimetic provided equivalent protection of PT cells from both diabetic and control rats. Despite the greater susceptibility to oxidative and mitochondrial injury, both cytoplasmic and mitochondrial glutathione concentrations were markedly higher in PT cells from diabetic rats, suggesting an upregulation of antioxidant processes in diabetic kidney. These results support the hypothesis that primary cultures of PT cells from diabetic rats are a valid model in which to study renal cellular function in the diabetic state.

  4. A hybrid substratum for primary hepatocyte culture that enhances hepatic functionality with low serum dependency.

    PubMed

    Meng, Qingyuan; Tao, Chunsheng; Qiu, Zhiye; Akaike, Toshihiro; Cui, Fuzhai; Wang, Xiumei

    2015-01-01

    Cell culture systems have proven to be crucial for the in vitro maintenance of primary hepatocytes and the preservation of hepatic functional expression at a high level. A poly-(N-p-vinylbenzyl-4-O-β-D-galactopyranosyl-D-gluconamide) matrix can recognize cells and promote liver function in a spheroid structure because of a specific galactose-asialoglycoprotein receptor interaction. Meanwhile, a fusion protein, E-cadherin-Fc, when incubated with various cells, has shown an enhancing effect on cellular viability and metabolism. Therefore, a hybrid substratum was developed for biomedical applications by using both of these materials to combine their advantages for primary hepatocyte cultures. The isolated cells showed a monolayer aggregate morphology on the coimmobilized surface and displayed higher functional expression than cells on traditional matrices. Furthermore, the hybrid system, in which the highest levels of cell adhesion and hepatocellular metabolism were achieved with the addition of 1% fetal bovine serum, showed a lower serum dependency than the collagen/gelatin-coated surface. Accordingly, this substrate may attenuate the negative effects of serum and further contribute to establishing a defined culture system for primary hepatocytes.

  5. Effects of the polymeric niche on neural stem cell characteristics during primary culturing.

    PubMed

    Haubenwallner, Stefan; Katschnig, Matthias; Fasching, Ulrike; Patz, Silke; Trattnig, Christa; Andraschek, Natascha; Grünbacher, Gerda; Absenger, Markus; Laske, Stephan; Holzer, Clemens; Balika, Werner; Wagner, Manuela; Schäfer, Ute

    2014-05-01

    The polymeric niche encountered by cells during primary culturing can affect cell fate. However, most cell types are primarily propagated on polystyrene (PS). A cell type specific screening for optimal primary culture polymers particularly for regenerative approaches seems inevitable. The effect of physical and chemical properties of treated (corona, oxygen/nitrogen plasma) and untreated cyclic olefin polymer (COP), polymethymethacrylate (PMMA), PP, PLA, PS, PC on neuronal stem cell characteristics was analyzed. Our comprehensive approach revealed plasma treated COP and PMMA as optimal polymers for primary neuronal stem cell culturing and propagation. An increase in the number of NT2/D1 cells with pronounced adhesion, metabolic activities and augmented expression of neural precursor markers was associated to the plasma treatment of surfaces of COP and PMMA with nitrogen or oxygen, respectively. A shift towards large cell sizes at stable surface area/volume ratios that might promote the observed increase in metabolic activities and distinct modulations in F-actin arrangements seem to be primarily mediated by the plasma treatment of surfaces. These results indicate that the polymeric niche has a distinct impact on various cell characteristics. The selection of distinct polymers and the controlled design of an optimized polymer microenvironment might thereby be an effective tool to promote essential cell characteristics for subsequent approaches.

  6. Toxic effects of anabolic-androgenic steroids in primary rat hepatic cell cultures.

    PubMed

    Welder, A A; Robertson, J W; Melchert, R B

    1995-08-01

    Hepatic complications in athletes and bodybuilders after abusing anabolic-androgenic steroids (AAS) have been reported. Hepatic injury, including cholestasis, peliosis hepatis, hyperplasia, and tumors, have been attributed to abuse of the 17 alpha-alkylated AAS. Some of these pathological conditions have been reversed when individuals were converted to nonalkylated AAS regimens. The purpose of this study was to determine and compare the direct toxic effects of commonly abused AAS (both 17 alpha-alkylated and nonalkylated) in primary hepatic cell cultures. Primary cultures, established from 60-day-old Sprague-Dawley rats, were exposed to doses of 1 x 10(-8), 1 x 10(-6), and 1 x 10(-4)M 19-nortestosterone, fluoxymesterone, testosterone cypionate, stanozolol, danazol, oxymetholone, testosterone, estradiol, and methyltestosterone for 1, 4, and 24 hr. Lactate dehydrogenase (LDH) release, neutral red (NR) retention, and glutathione (GSH) depletion were evaluated to determine plasma membrane damage, cell viability, and possible oxidative injury, respectively. Those cultures exposed to the 17 alpha-alkylated AAS, methyltestosterone and stanozolol, at doses of 1 x 10(-4) M for 24 hr and the 17 alpha-alkylated AAS, oxymetholone, at 1 x 10(-4) M for 4 and 24 hr showed significant increased in LDH release and decreases in NR retention while there were no significant differences with the nonalkylated steroids (testosterone cypionate, 19-nortestosterone, testosterone, and estradiol). GSH depletion was evaluated in cultures treated with 1 x 10(-8), 1 x 10(-6), and 1 x 10(-4) M concentrations of methyltestosterone, stanozolol, and oxymetholone for 1, 2, 4, and 6 hr. Cultures exposed to 1 x 10(-4) M oxymetholone were significantly depleted of GSH at 2, 4, and 6 hr; cultures exposed to 1 x 10(-4) M methyltestosterone were significantly depleted of GSH at 4 and 6 hr; and cultures exposed to stanozolol were not significantly depleted of GSH at any of the time periods tested. These

  7. Rotenone induces cell death in primary dopaminergic culture by increasing ROS production and inhibiting mitochondrial respiration.

    PubMed

    Radad, Khaled; Rausch, Wolf-Dieter; Gille, Gabriele

    2006-09-01

    Although the definite etiology of Parkinson's disease is still unclear, increasing evidence has suggested an important role for environmental factors such as exposure to pesticides in increasing the risk of developing Parkinson's disease. In the present study, primary cultures prepared from embryonic mouse mesencephala were applied to investigate the toxic effects and underlying mechanisms of rotenone-induced neuronal cell death relevant to Parkinson's disease. Results revealed that rotenone destroyed dopaminergic neurons in a dose- and time-dependent manner. Consistent with the cytotoxic effect of rotenone as evidenced by dopaminergic cell loss, it significantly increased the release of lactate dehydrogenase into the culture medium, the number of necrotic cells in the culture and the number of nuclei showing apoptotic features. Rotenone exerted toxicity by decreasing the mitochondrial membrane potential, increasing reactive oxygen species production and shifting respiration to a more anaerobic state.

  8. Harvesting Human Prostate Tissue Material and Culturing Primary Prostate Epithelial Cells.

    PubMed

    Frame, Fiona M; Pellacani, Davide; Collins, Anne T; Maitland, Norman J

    2016-01-01

    In order to fully explore the biology of a complex solid tumor such as prostate cancer, it is desirable to work with patient tissue. Only by working with cells from a tissue can we take into account patient variability and tumor heterogeneity. Cell lines have long been regarded as the workhorse of cancer research and it could be argued that they are of most use when considered within a panel of cell lines, thus taking into account specified mutations and variations in phenotype between different cell lines. However, often very different results are obtained when comparing cell lines to primary cells cultured from tissue. It stands to reason that cells cultured from patient tissue represents a close-to-patient model that should and does produce clinically relevant data. This chapter aims to illustrate the methods of processing, storing and culturing cells from prostate tissue, with a description of potential uses.

  9. Primary Retinal Cultures as a Tool for Modeling Diabetic Retinopathy: An Overview

    PubMed Central

    Varano, Monica; Mallozzi, Cinzia; Gaddini, Lucia; Formisano, Giuseppe; Pricci, Flavia

    2015-01-01

    Experimental models of diabetic retinopathy (DR) have had a crucial role in the comprehension of the pathophysiology of the disease and the identification of new therapeutic strategies. Most of these studies have been conducted in vivo, in animal models. However, a significant contribution has also been provided by studies on retinal cultures, especially regarding the effects of the potentially toxic components of the diabetic milieu on retinal cell homeostasis, the characterization of the mechanisms on the basis of retinal damage, and the identification of potentially protective molecules. In this review, we highlight the contribution given by primary retinal cultures to the study of DR, focusing on early neuroglial impairment. We also speculate on possible themes into which studies based on retinal cell cultures could provide deeper insight. PMID:25688355

  10. Aragonite crystallization in primary cell cultures of multicellular isolates from a hard coral, Pocillopora damicornis.

    PubMed

    Domart-Coulon, I J; Elbert, D C; Scully, E P; Calimlim, P S; Ostrander, G K

    2001-10-09

    The foundation of marine coral reef ecosystems is calcium carbonate accumulated primarily by the action of hard corals (Coelenterata: Anthozoa: Scleractinia). Colonial hard coral polyps cover the surface of the reef and deposit calcium carbonate as the aragonite polymorph, stabilized into a continuous calcareous skeleton. Scleractinian coral skeleton composition and architecture are well documented; however, the cellular mechanisms of calcification are poorly understood. There is little information on the nature of the coral cell types involved or their cooperation in biocalcification. We report aragonite crystallization in primary cell cultures of a hard coral, Pocillopora damicornis. Cells of apical coral colony fragments were isolated by spontaneous in vitro dissociation. Single dissociated cell types were separated by density in a discontinuous Percoll gradient. Primary cell cultures displayed a transient increase in alkaline phosphatase (ALP) activity, to the level observed in intact corals. In adherent multicellular isolate cultures, enzyme activation was followed by precipitation of aragonite. Modification of the ionic formulation of the medium prolonged maintenance of isolates, delayed ALP activation, and delayed aragonite precipitation. These results demonstrate that in vitro crystallization of aragonite in coral cell cultures is possible, and provides an innovative approach to investigate reef-building coral calcification at the cellular level.

  11. Aragonite crystallization in primary cell cultures of multicellular isolates from a hard coral, Pocillopora damicornis

    PubMed Central

    Domart-Coulon, Isabelle J.; Elbert, David C.; Scully, Erik P.; Calimlim, Precilia S.; Ostrander, Gary K.

    2001-01-01

    The foundation of marine coral reef ecosystems is calcium carbonate accumulated primarily by the action of hard corals (Coelenterata: Anthozoa: Scleractinia). Colonial hard coral polyps cover the surface of the reef and deposit calcium carbonate as the aragonite polymorph, stabilized into a continuous calcareous skeleton. Scleractinian coral skeleton composition and architecture are well documented; however, the cellular mechanisms of calcification are poorly understood. There is little information on the nature of the coral cell types involved or their cooperation in biocalcification. We report aragonite crystallization in primary cell cultures of a hard coral, Pocillopora damicornis. Cells of apical coral colony fragments were isolated by spontaneous in vitro dissociation. Single dissociated cell types were separated by density in a discontinuous Percoll gradient. Primary cell cultures displayed a transient increase in alkaline phosphatase (ALP) activity, to the level observed in intact corals. In adherent multicellular isolate cultures, enzyme activation was followed by precipitation of aragonite. Modification of the ionic formulation of the medium prolonged maintenance of isolates, delayed ALP activation, and delayed aragonite precipitation. These results demonstrate that in vitro crystallization of aragonite in coral cell cultures is possible, and provides an innovative approach to investigate reef-building coral calcification at the cellular level. PMID:11593000

  12. Culture-based identification of pigmented Porphyromonas and Prevotella species in primary endodontic infections

    PubMed Central

    Rajaram, Anuradha; Kotrashetti, Vijayalakshmi S.; Somannavar, Pradeep D.; Ingalagi, Preeti; Bhat, Kishore

    2016-01-01

    Background. The most common species isolated from primary endodontic infections are black-pigmented bacteria. These species are implicated in apical abscess formation due to their proteolytic activity and are fastidious in nature. Therefore, the present study was carried out to evaluate the presence and identification of various pigmented Porphyromonas and Prevotella species in the infected root canal through culture-based techniques. Methods. Thirty-one patients with primary endodontic infections were selected. Using sterile paper points, samples were collected from the root canals after access opening and prior to obturation, which were cultured using blood and kanamycin blood agar. Subsequently, biochemical test was used to identify the species and the results were analyzed using percentage comparison analysis, McNemar and chi-squared tests, Wilcoxon match pair test and paired t-test. Results. Out of 31 samples 26 were positive for black-pigmented organisms; the predominantly isolated species were Prevotella followed by Porphyromonas. In Porphyromonas only P. gingivalis was isolated. One of the interesting features was isolation of P. gingivalis through culture, which is otherwise very difficult to isolate through culture. Conclusion. The presence of Prevotella and Porphyromonas species suggests that a significant role is played by these organisms in the pathogenesis of endodontic infections. PMID:27651878

  13. Photodynamic therapy (PDT) of endometrium primary cultures serving as an in-vitro model for endometriosis

    NASA Astrophysics Data System (ADS)

    Herter, Wiebke; Viereck, Volker; Keckstein, J.; Steiner, Rudolf W.; Rueck, Angelika C.

    1994-05-01

    As a new treatment model for endometriosis, photodynamic therapy (PDT) was applied to endometrium cultures. Endometriosis is a benign disease. Therefore primary cultures were used instead of cell lines. Endometrium is composed of epithelial and stromal cells which can also be found in primary culture. While stromal cells take a polygonal shape in culture, epithelial cells form cell colonies. PSIII (Photasan III), which is similar to hematorporphyrin derivate (HpD), meso-tetra (4-sulfonatophenyl) porphyrin (TPPS4), which posses a high fluorescence quantum yield and may be useful in fluorescence diagnosis of subtle endometriotic spots, and methylene blue (MB), a vital dye with phototoxic properties, were used as photosensitizers. Different sensitizer concentrations and incubation times were applied. The highest phototoxicity was observed for PSIII; TPPS4 and MB were less phototoxic. We compared our results with the sensitivity of cell lines described in the literature. The necessary irradiation to destroy stromal cells was relatively high but still in the same dimension as for cell lines. However they were even more sensitive than epithelial cells. This was true for all sensitizers used.

  14. Photodynamic treatment (PDT) of endometrium primary cultures serving as an in-vitro-model for endometriosis

    NASA Astrophysics Data System (ADS)

    Werter, Wiebke; Viereck, Volker; Keckstein, J.; Steiner, Rudolf W.; Rueck, Angelika C.

    1994-05-01

    As a new treatment model for endometriosis, photodynamic therapy (PDT) was applied to endometrium cultures. Endometriosis is a benign disease. Therefore primary cultures were used instead of cell lines. Endometrium is composed of epithelial and stromal cells which can also be found in primary culture. While stromal cells take a polygonal shape in culture, epithelial cells form cell colonies. PSIII (Photasan III), which is similar to hematorporphyrin derivate (HpD), meso-tetra (4-sulfonatophenyl) porphyrin (TPPS4), which posses a high fluorescence quantum yield and may be useful in fluorescence diagnosis of subtle endometriotic spots, and methylene blue (MB), a vital dye with phototoxic properties, were used as photosensitizers. Different sensitizer concentrations and incubation times were applied. The highest phototoxicity was observed for PSIII; TPPS4 and MB were less phototoxic. We compared our results with the sensitivity of cell lines described in the literature. The necessary irradiation to destroy stromal cells was relatively high but still in the same dimension as for cell lines. However they were even more sensitive than epithelial cells. This was true for all sensitizers used.

  15. Protective Effect of Edaravone against Carbon Monoxide Induced Apoptosis in Rat Primary Cultured Astrocytes

    PubMed Central

    Xu, Xiaodan; Zhang, Hong; Wang, Ke; Tu, Tao

    2017-01-01

    Objective. To observe the protective effect of edaravone (Eda) on astrocytes after prolonged exposure to carbon monoxide (CO) and further to investigate the potential mechanisms of Eda against CO-induced apoptosis. Methods. The rat primary cultured astrocytes were cultured in vitro and exposed to 1% CO for 24 h after being cultured with different concentrations of Eda. MTT assay was used to detect the cytotoxicity of CO. Flow cytometry was used to detect the apoptosis rate, membrane potential of mitochondria, and ROS level. The mRNA and protein expressions of Bcl-2, Bax, and caspase-3 were assessed by real-time PCR and Western blotting analysis, respectively. Results. Eda can significantly suppress cytotoxicity of CO, and it can significantly increase membrane potential of mitochondria and Bcl-2 expressions and significantly suppress the apoptosis rate, ROS level, Bax, and caspase-3 expressions. Conclusion. Eda protects against CO-induced apoptosis in rat primary cultured astrocytes through decreasing ROS production and subsequently inhibiting mitochondrial apoptosis pathway. PMID:28261501

  16. E-Cigarette Affects the Metabolome of Primary Normal Human Bronchial Epithelial Cells.

    PubMed

    Aug, Argo; Altraja, Siiri; Kilk, Kalle; Porosk, Rando; Soomets, Ursel; Altraja, Alan

    2015-01-01

    E-cigarettes are widely believed to be safer than conventional cigarettes and have been even suggested as aids for smoking cessation. However, while reasonable with some regards, this judgment is not yet supported by adequate biomedical research data. Since bronchial epithelial cells are the immediate target of inhaled toxicants, we hypothesized that exposure to e-cigarettes may affect the metabolome of human bronchial epithelial cells (HBEC) and that the changes are, at least in part, induced by oxidant-driven mechanisms. Therefore, we evaluated the effect of e-cigarette liquid (ECL) on the metabolome of HBEC and examined the potency of antioxidants to protect the cells. We assessed the changes of the intracellular metabolome upon treatment with ECL in comparison of the effect of cigarette smoke condensate (CSC) with mass spectrometry and principal component analysis on air-liquid interface model of normal HBEC. Thereafter, we evaluated the capability of the novel antioxidant tetrapeptide O-methyl-l-tyrosinyl-γ-l-glutamyl-l-cysteinylglycine (UPF1) to attenuate the effect of ECL. ECL caused a significant shift in the metabolome that gradually gained its maximum by the 5th hour and receded by the 7th hour. A second alteration followed at the 13th hour. Treatment with CSC caused a significant initial shift already by the 1st hour. ECL, but not CSC, significantly increased the concentrations of arginine, histidine, and xanthine. ECL, in parallel with CSC, increased the content of adenosine diphosphate and decreased that of three lipid species from the phosphatidylcholine family. UPF1 partially counteracted the ECL-induced deviations, UPF1's maximum effect occurred at the 5th hour. The data support our hypothesis that ECL profoundly alters the metabolome of HBEC in a manner, which is comparable and partially overlapping with the effect of CSC. Hence, our results do not support the concept of harmlessness of e-cigarettes.

  17. Exploring processes of organization of normal and neoplastic epithelial tissues in gradient culture.

    PubMed

    Leighton, J

    1994-09-01

    The biology of animal cells in culture is often studied in individual cells or in sheets of cells. The relevance of such studies to the intact animal is unclear, since the spatial conditions encountered by cells in animals is one of dense three-dimensional masses of cells, with limits to migration, and with gradients both of diffusion of metabolites and of morphologic maturation. These spatial requisites have gradually been met in culture. A brief account describes sponge matrix culture for three-dimensional growth and unilaminar, bilaminar, and radial histophysiologic gradient cultures. Some of the common neoplastic abnormalities of surface epithelial tissues are considered. Proposals for investigating the histokinetic mechanisms regulating some epithelial tissue processes are suggested. In the most recent development of gradient culture methods, a thin permeable transparent collagen membrane is intrinsically strengthened by producing a waffle membrane pattern for histophysiologic gradient culture.

  18. Primary cultures of human colon cancer as a model to study cancer stem cells.

    PubMed

    Koshkin, Sergey; Danilova, Anna; Raskin, Grigory; Petrov, Nikolai; Bajenova, Olga; O'Brien, Stephen J; Tomilin, Alexey; Tolkunova, Elena

    2016-09-01

    The principal cause of death in cancer involves tumor progression and metastasis. Since only a small proportion of the primary tumor cells, cancer stem cells (CSCs), which are the most aggressive, have the capacity to metastasize and display properties of stem cells, it is imperative to characterize the gene expression of diagnostic markers and to evaluate the drug sensitivity in the CSCs themselves. Here, we have examined the key genes that are involved in the progression of colorectal cancer and are expressed in cancer stem cells. Primary cultures of colorectal cancer cells from a patient's tumors were studied using the flow cytometry and cytological methods. We have evaluated the clinical and stem cell marker expression in these cells, their resistance to 5-fluorouracil and irinotecan, and the ability of cells to form tumors in mice. The data shows the role of stem cell marker Oct4 in the resistance of primary colorectal cancer tumor cells to 5-fluorouracil.

  19. Primary culture of secretagogue-responsive parietal cells from rabbit gastric mucosa

    SciTech Connect

    Chew, C.S.; Ljungstroem, M.S.; Smolka, A.; Brown, M.R.

    1989-01-01

    A new procedure for isolation and primary culture of gastric parietal cells is described. Parietal cells from rabbit gastric mucosa are enriched to greater than 95% purity by combining a Nycodenz gradient separation with centrifugal elutriation. Cells are plated on the basement membrane matrix, Matrigel, and maintained in culture for at least 1 wk. Parietal cells cultured in this manner remain differentiated, cross-react with monoclonal H+-K+-ATPase antibodies, and respond to histamine, gastrin, and cholinergic stimulation with increased acid production as measured by accumulation of the weak base, (/sup 14/C)aminopyrine. When stimulated, cultured cells undergo ultrastructural changes in which intracellular canaliculi expand and numerous microvilli are observed. These ultrastructural changes are similar to those previously found to occur in vivo and in acutely isolated parietal cells. Morphological transformations in living cells can also be observed with differential interference contrast optics in the light microscope. After histamine stimulation, intracellular canaliculi gradually expand to form large vacuolar spaces. When the H2 receptor antagonist, cimetidine, is added to histamine-stimulated cells, these vacuoles gradually disappear. The ability to maintain hormonally responsive parietal cells in primary culture should make it possible to study direct, long-term effects of a variety of agonists and antagonists on parietal cell secretory-related activity. These cultured cells should also prove to be useful for the study of calcium transients, ion fluxes, and intracellular pH as related to acid secretion in single cells, particularly since morphological transformations can be used to monitor physiological responses at the same time within the same cell.

  20. Senescence Process in Primary Wilms' Tumor Cell Culture Induced by p53 Independent p21 Expression

    PubMed Central

    Theerakitthanakul, Korkiat; Saetang, Jirakrit; Kruatong, Jirasak; Graidist, Potchanapond; Raungrut, Pritsana; Kayasut, Kanita; Sangkhathat, Surasak

    2016-01-01

    Wilms tumor (WT) is an embryonal tumor occurring in developing kidney tissue. WT cells showing invasive cancer characteristics, also retain renal stem cell behaviours. In-vitro culture of WT is hampered by limited replicative potential. This study aimed to establish a longterm culture of WT cells to enable the study of molecular events to attempt to explain its cellular senescence. Methods: Primary cell cultures from fresh WT tumor specimen were established. Of 5 cultures tried, only 1 could be propagated for more than 7 passages. One culture, identified as PSU-SK-1, could be maintained > 35 passages and was then subjected to molecular characterization and evaluation for cancer characteristics. The cells consistently harbored concomitant mutations of CTNNB1 (Ser45Pro) and WT1 (Arg413Stop) thorough the cultivation. On Transwell invasion assays, the cells exhibited migration and invasion at 55% and 27% capability of the lung cancer cells, A549. On gelatin zymography, PSU-SK-1 showed high expression of the matrix metaloproteinase. The cells exhibited continuous proliferation with 24-hour doubling time until passages 28-30 when the growth slowed, showing increased cell size, retention of cells in G1/S proportion and positive β-galactosidase staining. As with those evidence of senescence in advanced cell passages, expression of p21 and cyclin D1 increased when the expression of β-catenin and its downstream protein, TCF, declined. There was also loss-of-expression of p53 in this cell line. In conclusion, cellular senescence was responsible for limited proliferation in the primary culture of WT, which was also associated with increased expression of p21 and was independent of p53 expression. Decreased activation of the Wnt signalling might explain the induction of p21 expression. PMID:27698927

  1. Excitability of the Primary Motor Cortex Increases More Strongly with Slow- than with Normal-Speed Presentation of Actions

    PubMed Central

    Moriuchi, Takefumi; Iso, Naoki; Sagari, Akira; Ogahara, Kakuya; Kitajima, Eiji; Tanaka, Koji; Tabira, Takayuki; Higashi, Toshio

    2014-01-01

    Introduction The aim of the present study was to investigate how the speed of observed action affects the excitability of the primary motor cortex (M1), as assessed by the size of motor evoked potentials (MEPs) induced by transcranial magnetic stimulation (TMS). Methods Eighteen healthy subjects watched a video clip of a person catching a ball, played at three different speeds (normal-, half-, and quarter-speed). MEPs were induced by TMS when the model's hand had opened to the widest extent just before catching the ball (“open”) and when the model had just caught the ball (“catch”). These two events were locked to specific frames of the video clip (“phases”), rather than occurring at specific absolute times, so that they could easily be compared across different speeds. MEPs were recorded from the thenar (TH) and abductor digiti minimi (ADM) muscles of the right hand. Results The MEP amplitudes were higher when the subjects watched the video clip at low speed than when they watched the clip at normal speed. A repeated-measures ANOVA, with the factor VIDEO-SPEED, showed significant main effects. Bonferroni's post hoc test showed that the following MEP amplitude differences were significant: TH, normal vs. quarter; ADM, normal vs. half; and ADM, normal vs. quarter. Paired t-tests showed that the significant MEP amplitude differences between TMS phases under each speed condition were TH, “catch” higher than “open” at quarter speed; ADM, “catch” higher than “open” at half speed. Conclusions These results indicate that the excitability of M1 was higher when the observed action was played at low speed. Our findings suggest that the action observation system became more active when the subjects observed the video clip at low speed, because the subjects could then recognize the elements of action and intention in others. PMID:25479161

  2. The implementation of a social constructivist approach in primary science education in Confucian heritage culture: the case of Vietnam

    NASA Astrophysics Data System (ADS)

    Hằng, Ngô Vũ Thu; Meijer, Marijn Roland; Bulte, Astrid M. W.; Pilot, Albert

    2015-09-01

    Social constructivism has been increasingly studied and implemented in science school education. Nevertheless, there is a lack of holistic studies on the implementation of social constructivist approach in primary science education in Confucian heritage culture. This study aims to determine to what extent a social constructivist approach is implemented in primary science education in Confucian heritage culture and to give explanations for the implementation from a cultural perspective. Findings reveal that in Confucian heritage culture a social constructivist approach has so far not implemented well in primary science education. The implementation has been considerably influenced by Confucian heritage culture, which has characteristics divergent from and aligning with those of social constructivism. This study indicates a need for design-based research on social constructivism-based science curriculum for Confucian heritage culture.

  3. Changes in adipose tissue stromal-vascular cells in primary culture due to porcine sera

    SciTech Connect

    Jewell, D.E.; Hausman, G.J.

    1986-03-01

    This study was conducted to determine the response of rat stromal-vascular cells to pig sea. Sera were collected from unselected contemporary (lean) and high backfat thickness selected (obese) pigs. Sera from obese pigs were collected either by exsanguination or cannulation. sera from lean pigs during the growing phase (45 kg) and the fattening phase (100-110 kg) were collected. Stromal-vascular cells derived rom rat inguinal tissue were cultured on either 25 cm/sup 2/ flasks, collagen-coated coverslips or petri dishes. Cell proliferation was measured by (/sup 3/H)-thymidine incorporation during the fourth day of culture. Coverslip cultures were used for histochemical analysis. Petri dish cultures were used for analysis of Sn-glycerol-3-phosphate dehydrogenase (GPDH) activity. All cells were plated for 24 hours in media containing 10 fetal bovine sera. Test media contained 2.5, 5.0, 10.0% sera. Sera from obese pigs increased GPDH activity and fat cell production when compared to the lean controls. The increased concentration of sera increased esterase activity and lipid as measured with oil red O. The sera from obese pigs collected at slaughter stimulated more fat cell production than obese sera collected by cannulation. These studies show there are adipogenic factors in obese pigs sera which promote fat cell development in primary cell culture.

  4. Primary Culture of Mycobacterium ulcerans from Human Tissue Specimens after Storage in Semisolid Transport Medium▿

    PubMed Central

    Eddyani, Miriam; Debacker, Martine; Martin, Anandi; Aguiar, Julia; Johnson, Christian R.; Uwizeye, Cécile; Fissette, Krista; Portaels, Françoise

    2008-01-01

    Tissue specimens collected from patients with clinically suspected Buruli ulcer treated in two Buruli ulcer treatment centers in Benin between 1998 and 2004 were placed in semisolid transport medium and transported at ambient temperature for microbiological analysis at the Institute of Tropical Medicine in Antwerp, Belgium. The impact of the delay before microbiological analysis on primary culture of Mycobacterium ulcerans was investigated. The length of storage in semisolid transport medium varied from 6 days to 26 weeks. Of the 1,273 tissue fragments positive for M. ulcerans DNA by an IS2404-specific PCR, 576 (45.2%) yielded positive culture results. The sensitivity of direct smear examination was 64.6% (822/1,273 tissue fragments). The median time required to obtain a positive culture result was 11 weeks. Positive cultures were obtained even from samples kept for more than 2 months at ambient temperatures. Moreover, there was no reduction in the viability of M. ulcerans, as detected by culture, when specimens remained in semisolid transport medium for long periods of time (up to 26 weeks). We can conclude that the method with semisolid transport medium is very robust for clinical specimens from patients with Buruli ulcer that, due to circumstances, cannot be analyzed in a timely manner. This transport medium is thus very useful for the confirmation of a diagnosis of Buruli ulcer with specimens collected in the field. PMID:17989199

  5. Primary possum macrophage cultures support the growth of a nidovirus associated with wobbly possum disease.

    PubMed

    Giles, Julia C; Perrott, Matthew R; Dunowska, Magdalena

    2015-09-15

    The objective of the study was to establish a system for isolation of a recently described, thus far uncultured, marsupial nidovirus associated with a neurological disease of possums, termed wobbly possum disease (WPD). Primary cultures of possum macrophages were established from livers of adult Australian brushtail possums (Trichosurus vulpecula). High viral copy numbers (up to 6.9×10(8)/mL of cell lysate) were detected in infected cell culture lysates from up to the 5th passage of the virus, indicating that the putative WPD virus (WPDV) was replicating in cultured cells. A purified virus stock with a density of 1.09 g/mL was prepared using iodixanol density gradient ultracentrifugation. Virus-like particles approximately 60 nm in diameter were observed using electron microscopy in negatively stained preparations of the purified virus. The one-step growth curve of WPDV in macrophage cultures showed the highest increase in intracellular viral RNA between 6 and 12h post-infection. Maximum levels of cell-associated viral RNA were detected at 24h post-infection, followed by a decline. Levels of extracellular RNA increased starting at 9h post-infection, with maximum levels detected at 48 h post-infection. The establishment of the in vitro system to culture WPDV will facilitate further characterisation of this novel nidovirus.

  6. Primary liver cells cultured on carbon nanotube substrates for liver tissue engineering and drug discovery applications.

    PubMed

    Che Abdullah, Che Azurahanim; Azad, Chihye Lewis; Ovalle-Robles, Raquel; Fang, Shaoli; Lima, Marcio D; Lepró, Xavier; Collins, Steve; Baughman, Ray H; Dalton, Alan B; Plant, Nick J; Sear, Richard P

    2014-07-09

    Here, we explore the use of two- and three-dimensional scaffolds of multiwalled-carbon nanotubes (MWNTs) for hepatocyte cell culture. Our objective is to study the use of these scaffolds in liver tissue engineering and drug discovery. In our experiments, primary rat hepatocytes, the parenchymal (main functional) cell type in the liver, were cultured on aligned nanogrooved MWNT sheets, MWNT yarns, or standard 2-dimensional culture conditions as a control. We find comparable cell viability between all three culture conditions but enhanced production of the hepatocyte-specific marker albumin for cells cultured on MWNTs. The basal activity of two clinically relevant cytochrome P450 enzymes, CYP1A2 and CYP3A4, are similar on all substrates, but we find enhanced induction of CYP1A2 for cells on the MWNT sheets. Our data thus supports the use of these substrates for applications including tissue engineering and enhancing liver-specific functions, as well as in in vitro model systems with enhanced predictive capability in drug discovery and development.

  7. Epithelial-mesenchymal transitions during cell culture of primary thyroid tumors?

    PubMed

    Herrmann, M E; Trevor, K T

    1993-04-01

    Fibroblast contamination of epithelial tumor cell cultures is of great concern when examining tumor cells in vitro for specific biochemical and cytogenetic changes. The observations of normal karyotypes in thyroid tumor cell cultures have raised the concern of whether residual tissue fibroblasts might obscure the cytogenetic analysis of transformed epithelial cells. We have characterized early passaged thyroid tumor cells to examine the proportions of epithelial and fibroblastic cell types. Cells were analyzed by immunocytology using antibodies recognizing the thyroid prohormone thyroglobulin, epithelial cytokeratins, and vimentin, a mesenchyme marker. Tumors consisted of one follicular adenoma and five papillary carcinomas. When examined by day 15 in culture, all cells contained filaments composed of vimentin, which most likely represents an adaptation to culture conditions. Double immunofluorescence staining for thyroglobulin and cytokeratin revealed the presence of not only epithelial but also spindle-like fibroblastoid cells possessing thyroid epithelial cell markers. The results suggest that in thyroid tumor cultures there is a unique cell type intermediate between epithelial and mesenchyme phenotypes that must be considered when performing cytogenetic analysis.

  8. The effect of anabolic-androgenic steroids on primary myocardial cell cultures.

    PubMed

    Melchert, R B; Herron, T J; Welder, A A

    1992-02-01

    Although recent case reports suggest that anabolic-androgenic steroids may be directly injurious to the cardiovascular system, the direct myocardial cellular consequences of abuse of these drugs are not known. Therefore, the purpose of this study was to describe the concentration- and time-dependent effects of testosterone cypionate (TC), stanozolol (S), and fluoxymesterone (F) on primary myocardial cell cultures. Evaluation of drug effects were made in 4-d-old primary myocardial cell cultures obtained from 3- to 5-d-old Sprague-Dawley rats. The cultures were exposed to 1 x 10(-4) M, 1 x 10(-6) M, and 1 x 10(-8) M concentrations of TC, S, and F each for 1, 4, and 24 h. Cellular injury was evaluated by alterations in beating activity, induction of morphological alterations, lactate dehydrogenase (LDH) release, neutral red retention, and tetrazolium (MTT) formazan production. Significant alterations in beating activity were observed in the 1 x 10(-4) M TC group in which no beating activity was seen at 1, 4, and 24 h. Morphological integrity was disrupted for the 1 x 10(-4) M TC group at 24 h where destruction of the monolayer was observed. Unlike the cultures treated with the three concentrations of both S and F, significant LDH release was seen at 4 and 24 h with those cultures exposed to 1 x 10(-4) M TC. In the evaluation of neutral red retention, 1 x 10(-4) M TC at 24 h showed a significant decrease in ability to retain the dye.(ABSTRACT TRUNCATED AT 250 WORDS)

  9. Sugammadex, a Neuromuscular Blockade Reversal Agent, Causes Neuronal Apoptosis in Primary Cultures

    PubMed Central

    Palanca, José M.; Aguirre-Rueda, Diana; Granell, Manuel V.; Aldasoro, Martin; Garcia, Alma; Iradi, Antonio; Obrador, Elena; Mauricio, Maria Dolores; Vila, Jose; Gil-Bisquert, Anna; Valles, Soraya L.

    2013-01-01

    Sugammadex, a γ-cyclodextrin that encapsulates selectively steroidal neuromuscular blocking agents, such as rocuronium or vecuronium, has changed the face of clinical neuromuscular pharmacology. Sugammadex allows a rapid reversal of muscle paralysis. Sugammadex appears to be safe and well tolerated. Its blood-brain barrier penetration is poor (< 3% in rats), and thus no relevant central nervous toxicity is expected. However the blood brain barrier permeability can be altered under different conditions (i.e. neurodegenerative diseases, trauma, ischemia, infections, or immature nervous system). Using MTT, confocal microscopy, caspase-3 activity, cholesterol quantification and Western-blot we determine toxicity of Sugammadex in neurons in primary culture. Here we show that clinically relevant sugammadex concentrations cause apoptotic/necrosis neuron death in primary cultures. Studies on the underlying mechanism revealed that sugammadex-induced activation of mitochondria-dependent apoptosis associates with depletion of neuronal cholesterol levels. Furthermore SUG increase CytC, AIF, Smac/Diablo and CASP-3 protein expression in cells in culture. Potential association of SUG-induced alteration in cholesterol homeostasis with oxidative stress and apoptosis activation occurs. Furthermore, resistance/sensitivity to oxidative stress differs between neuronal cell types. PMID:23983586

  10. Dual signal transduction pathways activated by TSH receptors in rat primary tanycyte cultures.

    PubMed

    Bolborea, Matei; Helfer, Gisela; Ebling, Francis J P; Barrett, Perry

    2015-06-01

    Tanycytes play multiple roles in hypothalamic functions, including sensing peripheral nutrients and metabolic hormones, regulating neurosecretion and mediating seasonal cycles of reproduction and metabolic physiology. This last function reflects the expression of TSH receptors in tanycytes, which detect photoperiod-regulated changes in TSH secretion from the neighbouring pars tuberalis. The present overall aim was to determine the signal transduction pathway by which TSH signals in tanycytes. Expression of the TSH receptor in tanycytes of 10-day-old Sprague Dawley rats was observed by in situ hybridisation. Primary ependymal cell cultures prepared from 10-day-old rats were found by immunohistochemistry to express vimentin but not GFAP and by PCR to express mRNA for Dio2, Gpr50, Darpp-32 and Tsh receptors that are characteristic of tanycytes. Treatment of primary tanycyte/ependymal cultures with TSH (100  IU/l) increased cAMP as assessed by ELISA and induced a cAMP-independent increase in the phosphorylation of ERK1/2 as assessed by western blot analysis. Furthermore, TSH (100  IU/l) stimulated a 2.17-fold increase in Dio2 mRNA expression. We conclude that TSH signal transduction in cultured tanycytes signals via Gαs to increase cAMP and via an alternative G protein to increase phosphorylation of ERK1/2.

  11. Androgen and estrogen receptors are present in primary cultures of human synovial macrophages.

    PubMed

    Cutolo, M; Accardo, S; Villaggio, B; Barone, A; Sulli, A; Coviello, D A; Carabbio, C; Felli, L; Miceli, D; Farruggio, R; Carruba, G; Castagnetta, L

    1996-02-01

    Macrophages, as antigen-processing and -presenting cells to T lymphocytes, play a key role in the immune system and are suspected to be target cells of the sex hormone-related dimorphism in the immune response peculiar to rheumatoid arthritis (RA) pathology. In the present study, the use of specific monoclonal antibodies revealed immunostaining for androgen and estrogen receptors in primary cultures of macrophages obtained from synovial tissues of patients affected by RA and controls without RA disease. Soluble and nuclear type I (high affinity, low capacity) and type II (lower affinity, greater capacity) sites of androgen or estrogen binding were detected in primary cultures of RA macrophages using radioligand binding assay. Higher levels of type I and type II estrogen receptor compared to those of androgen receptor were found, particularly in the soluble fraction; however, contrary to what was observed in whole synovial tissues, higher steroid receptor concentrations were found in the soluble than in the nuclear fraction of RA synovial macrophages. Binding affinities and receptor contents of cultured synovial macrophages were comparable to those previously reported in other well established sex hormone-responsive cells and tissues. Further, specific messenger ribonucleic acids for sex hormone receptors, encoding for a sequence of the DNA-binding domain of the receptor proteins were revealed by RT-PCR.

  12. [Use of hepatocytes in primary cultures to predict potential hepatocarcinogenicity of compounds

    PubMed

    Bieri, F.; Muakkassah-Kelly, S.; Bentley, P.

    1990-01-01

    Short-term tests for genotoxic activity will detect a variety of potential carcinogens. However, experience over the last few years has shown that many chemical carcinogens are not detected in these tests. A large proportion of these non-mutagenic (non-genotoxic) carcinogens induce tumors in the rodent liver after life-long feeding. One class of such carcinogens are the hypolipidemic drugs many of which induce a characteristic hepatomegaly upon subchronic treatment. Typically this liver enlargement is accompanied by increased mitotic activity and a proliferation of the hepatic peroxisome compartment. Results also suggest a correlation between the degree and the nature of the hepatomegalic response to a compound, and its hepatocarcinogenic potency, but it remains unclear whether the growth stimulation and/or the peroxisome proliferation is correlated with the carcinogenicity. Both, the induction of peroxisomal enzyme and of replicative DMA synthesis may be measured in primary cultures of adult rat hepatocytes following in vitro exposition. Moreover, the ability of the compounds tested to induce in vitro replicative DNA synthesis in primary cultures of adult rat hepatocytes correlated well with the potency of the hepatomegalic response induced in vivo in treated rodents. Consequently, studies using such cultures might be useful to characterize and possibly predict in vitro the hepatocarcinogenic potency of some new compounds. The early recognition of the possible carcinogenic property of a new substance might accordingly contribute to the reduction of a number of life-long studies.

  13. Differentiation of primary human submandibular gland cells cultured on basement membrane extract.

    PubMed

    Szlávik, Vanda; Szabó, Bálint; Vicsek, Tamás; Barabás, József; Bogdán, Sándor; Gresz, Veronika; Varga, Gábor; O'Connell, Brian; Vág, János

    2008-11-01

    There is no effective treatment for the loss of functional salivary tissue after irradiation for head and neck cancer or the autoimmune disease Sjögren's syndrome. One possible approach is the regeneration of salivary glands from stem cells. The present study aimed to investigate whether small pieces of human submandiblar gland tissue contain elements necessary for the reconstruction of salivary rudiments in vitro via acinar and ductal cell differentiation. Primary submandibular gland (primary total human salivary gland; PTHSG) cells were isolated from human tissue and cultured in vitro using a new method in which single cells form an expanding epithelial monolayer on plastic substrates. Differentiation, morphology, number, and organization of these cells were then followed on basement membrane extract (BME) using RNA quantitation (amylase, claudin-1 (CLN1), CLN3, kallikrein, vimentin), immunohistochemistry (amylase and occludin), viability assay, and videomicroscopy. On the surface of BME, PTHSG cells formed acinotubular structures within 24 h, did not proliferate, and stained for amylase. In cultures derived from half of the donors, the acinar markers amylase and CLN3 were upregulated. The PTHSG culture model suggests that human salivary gland may be capable of regeneration via reorganization and differentiation and that basement membrane components play a crucial role in the morphological and functional differentiation of salivary cells.

  14. Cerium oxide nanoparticles prevent apoptosis in primary cortical culture by stabilizing mitochondrial membrane potential.

    PubMed

    Arya, A; Sethy, N K; Das, M; Singh, S K; Das, A; Ujjain, S K; Sharma, R K; Sharma, M; Bhargava, K

    2014-07-01

    Cerium oxide nanoparticles (CNPs) of spherical shape have unique antioxidant capacity primarily due to alternating + 3 and + 4 oxidation states and crystal defects. Several studies revealed the protective efficacies of CNPs in cells and tissues against the oxidative damage. However, its effect on mitochondrial functioning, downstream effectors of radical burst and apoptosis remains unknown. In this study, we investigated whether CNPs treatment could protect the primary cortical cells from loss of mitochondrial membrane potential (Δψm) and Δψm-dependent cell death. CNPs with spherical morphology and size range 7-10 nm were synthesized and utilized at a concentration of 25 nM on primary neuronal culture challenged with 50 μM of hydrogen peroxide (H2O2). We showed that optimal dose of CNPs minimized ROS content of the cells and also curbed related surge in cellular calcium flux. Importantly, CNPs treatment prevented apoptotic loss of cell viability. Reduction in the apoptosis could be successfully attributed to the maintenance of Δψm and restoration of major redox equivalents NADH/NAD(+) ratio and cellular ATP. These findings, therefore, suggest possible route of CNPs protective efficacies in primary cortical culture.

  15. Cinematographic observations of growth cycles of Chlamydia trachomatis in primary cultures of human amniotic cells.

    PubMed Central

    Neeper, I D; Patton, D L; Kuo, C C

    1990-01-01

    Time-lapse cinematography was used to study the growth cycle of Chlamydia trachomatis in primary cell cultures of human amnion. Twelve preterm and twelve term placentas were obtained within 8 h of delivery, and epithelial cells were dissociated from the amniotic membranes by trypsinization and grown in Rose chambers. The epithelial nature of the cultured cells was documented by morphology and by immunofluorescence staining for cytoskeletal proteins, which matched the staining of intact amnion. With regular feedings, uninfected cultures remained healthy for up to 30 days. Confluent cultures (7 to 10 days) were infected with a genital strain (E/UW-5/CX) of C. trachomatis at 10(5) infectious units per chamber. Infections were done in culture medium without cycloheximide, which is often used to induce susceptibility of the cells. Between 66 and 90% of the cells were infected. Intracytoplasmic inclusions were visible by 18 h post infection (p.i.) and grew larger as the organisms inside multiplied. By 72 h p.i., the inclusions occupied the entire cytoplasm of the host cells. Further growth of the inclusions overdistended and ruptured the host cells on days 3 to 7. Cells not infected by the original inoculum became infected on day 5 or 6 p.i. by the chlamydial particles released from the ruptured cells. No amniotic cell was ever observed to survive the infection. The data presented support the hypothesis that amniotic epithelium is susceptible to infection and damage by C. trachomatis. This culture system provided detailed and dynamic observations of chlamydial infection under conditions more nearly physiologic than previously reported. Images PMID:2365450

  16. Utilization of supplemental methionine sources by primary cultures of chick hepatocytes

    SciTech Connect

    Dibner, J.J.

    1983-10-01

    Utilization of 2-hydroxy-4-(methylthio) butanoic acid (HMB) as a substrate for protein synthesis was studied by using primary cultures of chick liver cells. Cultures were prepared by enzymatic dissociation of livers from week old Hubbard broiler chicks and were maintained for 4 days under nonproliferative conditions. Hepatocyte differentiation was verified by using dexamethasone induction of tyrosine aminotransferase activity. Conversion of (14C)HMB to L-methionine was shown by chromatographic analysis of hepatocyte protein hydrolysate and incorporation into protein was proven by cycloheximide inhibition of synthesis. When incorporation of HMB was compared to that of DL-methionine (DLM) equimolar quantities of the two sources were found in liver cell protein. These results support, at a cellular level, the conclusion that HMB and DLM are biochemically equivalent sources of methionine for protein synthesis.

  17. Genetic basis of triticale breeding (x triticale). IV. Embryo culture for synthesizing primary hexaploid triticales

    SciTech Connect

    Gordei, I.A.; Khodortsova, L.F.

    1986-07-01

    Results are reported on enhancing the efficiency of embryo culture for synthesizing primary hexaploid triticales (AABBRR, 2n = 42). The antioxidant tomatoside has a positive effect on the reduction of progamous incompatibility of wheat with rye and increases the output of wheat-rye amphihaploids. It has been established that irradiation of embryos, cultured on nutrient medium, with helium-neon laser, increases significantly (P < 0.01) the regeneration frequency of the wheat-rye hybrid embryos. The highest frequency (40%) of amphidiploids was obtained by treating the plants with 0.15% colchicine through roots during the tillering phase. Hexaploid triticales from 11 cross combinations between tetraploid wheats (AABB, 2n = 28) and diploid rye (RR, 2n = 14) formed the initial material for breeding.

  18. Using homogeneous primary neuron cultures to study fundamental aspects of HSV-1 latency and reactivation.

    PubMed

    Kim, Ju Youn; Shiflett, Lora A; Linderman, Jessica A; Mohr, Ian; Wilson, Angus C

    2014-01-01

    We describe a primary neuronal culture system suitable for molecular characterization of herpes simplex virus type 1 (HSV-1) infection, latency, and reactivation. While several alternative models are available, including infections of live animal and explanted ganglia, these are complicated by the presence of multiple cell types, including immune cells, and difficulties in manipulating the neuronal environment. The highly pure neuron culture system described here can be readily manipulated and is ideal for molecular studies that focus exclusively on the relationship between the virus and host neuron, the fundamental unit of latency. As such it allows for detailed investigations of both viral and neuronal factors involved in the establishment and maintenance of HSV-1 latency and in viral reactivation induced by defined stimuli.

  19. Betulin elicits anti-cancer effects in tumour primary cultures and cell lines in vitro.

    PubMed

    Rzeski, Wojciech; Stepulak, Andrzej; Szymański, Marek; Juszczak, Małgorzata; Grabarska, Aneta; Sifringer, Marco; Kaczor, Józef; Kandefer-Szerszeń, Martyna

    2009-12-01

    Betulin is a pentacyclic triterpene found in many plant species, among others, in white birch bark. The aim of the study was in vitro characterization of the anticancer activity of betulin in a range of human tumour cell lines (neuroblastoma, rhabdomyosarcoma-medulloblastoma, glioma, thyroid, breast, lung and colon carcinoma, leukaemia and multiple myeloma), and in primary tumour cultures isolated from patients (ovarian carcinoma, cervical carcinoma and glioblastoma multiforme). In this study, we demonstrated a remarkable anti-proliferative effect of betulin in all tested tumour cell cultures. Neuroblastoma (SK-N-AS) and colon carcinoma (HT-29) were the most sensitive to the anti-proliferative effect of betulin. Furthermore, betulin altered tumour cells morphology, decreased their motility and induced apoptotic cell death. These findings demonstrate the anti-cancer potential of betulin and suggest that they may be applied as an adjunctive measure in cancer treatment.

  20. Immobilization of primary cultures of insulin-releasing human pancreatic cells.

    PubMed

    Mantovani, Marluce da Cunha; da Conceição, Mateus Meneghesso; Ferreira, Ari José Scattone; Labriola, Letícia; dos Santos, Patrícia Barros; Tonso, Aldo; Pereira, Carlos Augusto; El-Dorry, Hamza; Sogayar, Mari Cleide

    2009-01-01

    Transplantation of pancreatic islets isolated from organ donors constitutes a promising alternative treatment for type1 Diabetes, however, it is severely limited by the shortage of organ donors. Ex-vivo islet cell cultures appear as an attractive but still elusive approach for curing type 1 Diabetes. It has recently been shown that, even in the absence of fibrotic overgrowth, several factors, such as insufficient nutrition of the islet core, represent a major barrier for long-term survival of islets grafts. The use of immobilized dispersed cells may contribute to solve this problem due to conceivably easier nutritional and oxygen support to the cells.  Therefore, we set out to establish an immobilization method for primary cultures of human pancreatic cells by adsorption onto microcarriers (MCs). Dispersed human islets cells were seeded onto Cytodex1 microcarriers and cultured in bioreactors for up to eight days. The cell number increased and islet cells maintained their insulin secretion levels throughout the time period studied. Moreover, the cells also presented a tendency to cluster upon five days culturing.  Therefore, this procedure represents a useful tool for controlled studies on islet cells physiology and, also, for biotechnological applications.

  1. Claudins in a primary cultured puffer fish (Tetraodon nigroviridis) gill epithelium.

    PubMed

    Bui, Phuong; Kelly, Scott P

    2011-01-01

    A primary cultured gill epithelium from the model organism Tetraodon nigroviridis (spotted green puffer fish) has been developed for the study of claudin tight junction (TJ) proteins and their potential role in the regulation of paracellular permeability across the gills of fishes. The cultured preparation is composed of polygonal epithelial cells that exhibit TJ protein immunoreactivity around the periphery and develop a surface morphology of concentric apical microridges. There is an absence of cells exhibiting intense Na+-K+-ATPase immunoreactivity and taken together, these characteristics indicate that the epithelium is composed of gill pavement cells only. In Tetraodon, 52 genes encoding for claudin isoforms (Tncldn) have been identified and 32 of these genes are expressed in whole gill tissue. Of these genes, 12 are responsive to alterations in environmental salinity in vivo (Tncldn3a, -3c, -6, -8d, -10d, -10e, -11a, -23b, -27a, -27c, -32a, and -33b). All claudin isoforms found in whole gill tissue can be found in cultured pavement cell gill epithelia with the exception of Tncldn6, -10d, and -10e. The cultured preparation is suitable for studying the "molecular machinery" of TJ proteins in fish gill pavement cells.

  2. Effect of acrylamide-induced neurotoxicity in a primary astrocytes/microglial co-culture model.

    PubMed

    Zhao, Mengyao; Wang, Fu Sheng Lewis; Hu, Xiao Song; Chen, Fang; Chan, Hing Man

    2017-03-01

    Acrylamide (AA), is a common food contaminant generated by heat processing. Astrocytes and microglia are the two major glial cell types in the brain that play pivotal but different roles in maintaining optimal brain function. The objective of this study is to investigate the neurotoxicity of AA, using a primary astrocytes/microglia co-culture model. Co-cultural cells obtained from Balb/c mice were cultured and treated with 0-1.0mM AA for 24-96h. Cell viability, reactive oxygen species (ROS) generation, oxidative end produces formation and glutathione (GSH) levels were measured. The expression of nuclear-E2-related factor 2(Nrf2), and nuclear factor kappa-beta (NF-κB) and selected down-stream genes were measured. Results showed that AA treatment led toa dose-dependent toxicity. Oxidative stress was induced as indicated by an increase of ROS, a decrease of GSH levels, and an increase in the formation of 4-hydroxynonenal-adduct and 8-hydroxy-2-deoxyguanosine-adduct. Both Nrf2 and NF-κB pathway contributed to the initiation of oxidative stress but the timing of two factors was different. Nrf2 and its related downstream genes were activated earlier than that in NF-κB pathway. In conclusion, AA-induced neurotoxicity attribute to oxidative stress via Nrf2 and NF-κB pathway. Moreover, the co-culture cell model was proven to be a viable model to study AA neurotoxicity.

  3. Subretinal Pigment Epithelial Deposition of Drusen Components Including Hydroxyapatite in a Primary Cell Culture Model

    PubMed Central

    Pilgrim, Matthew G.; Lengyel, Imre; Lanzirotti, Antonio; Newville, Matt; Fearn, Sarah; Emri, Eszter; Knowles, Jonathan C.; Messinger, Jeffrey D.; Read, Russell W.; Guidry, Clyde; Curcio, Christine A.

    2017-01-01

    Purpose Extracellular deposits containing hydroxyapatite, lipids, proteins, and trace metals that form between the basal lamina of the RPE and the inner collagenous layer of Bruch's membrane are hallmarks of early AMD. We examined whether cultured RPE cells could produce extracellular deposits containing all of these molecular components. Methods Retinal pigment epithelium cells isolated from freshly enucleated porcine eyes were cultured on Transwell membranes for up to 6 months. Deposit composition and structure were characterized using light, fluorescence, and electron microscopy; synchrotron x-ray diffraction and x-ray fluorescence; secondary ion mass spectroscopy; and immunohistochemistry. Results Apparently functional primary RPE cells, when cultured on 10-μm-thick inserts with 0.4-μm-diameter pores, can produce sub-RPE deposits that contain hydroxyapatite, lipids, proteins, and trace elements, without outer segment supplementation, by 12 weeks. Conclusions The data suggest that sub-RPE deposit formation is initiated, and probably regulated, by the RPE, as well as the loss of permeability of the Bruch's membrane and choriocapillaris complex associated with age and early AMD. This cell culture model of early AMD lesions provides a novel system for testing new therapeutic interventions against sub-RPE deposit formation, an event occurring well in advance of the onset of vision loss. PMID:28146236

  4. Evaluating the Role of Viral Proteins in HIV-Mediated Neurotoxicity Using Primary Human Neuronal Cultures

    PubMed Central

    Rao, Vasudev R.; Eugenin, Eliseo A.; Prasad, Vinayaka R.

    2016-01-01

    Despite the inability of HIV-1 to infect neurons, over half of the HIV-1-infected population in the USA suffers from neurocognitive dysfunction. HIV-infected immune cells in the periphery enter the central nervous system by causing a breach in the blood–brain barrier. The damage to the neurons is mediated by viral and host toxic products released by activated and infected immune and glial cells. To evaluate the toxicity of any viral isolate, viral protein, or host inflammatory protein, we describe a protocol to assess the neuronal apoptosis and synaptic compromise in primary cultures of human neurons and astrocytes. PMID:26714725

  5. Evaluating the Role of Viral Proteins in HIV-Mediated Neurotoxicity Using Primary Human Neuronal Cultures.

    PubMed

    Rao, Vasudev R; Eugenin, Eliseo A; Prasad, Vinayaka R

    2016-01-01

    Despite the inability of HIV-1 to infect neurons, over half of the HIV-1-infected population in the USA suffers from neurocognitive dysfunction. HIV-infected immune cells in the periphery enter the central nervous system by causing a breach in the blood-brain barrier. The damage to the neurons is mediated by viral and host toxic products released by activated and infected immune and glial cells. To evaluate the toxicity of any viral isolate, viral protein, or host inflammatory protein, we describe a protocol to assess the neuronal apoptosis and synaptic compromise in primary cultures of human neurons and astrocytes.

  6. Primary culture and plasmid electroporation of the murine organ of Corti.

    PubMed

    Parker, Mark; Brugeaud, Aurore; Edge, Albert S B

    2010-02-04

    In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells. An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants. This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary

  7. Process cost and facility considerations in the selection of primary cell culture clarification technology.

    PubMed

    Felo, Michael; Christensen, Brandon; Higgins, John

    2013-01-01

    The bioreactor volume delineating the selection of primary clarification technology is not always easily defined. Development of a commercial scale process for the manufacture of therapeutic proteins requires scale-up from a few liters to thousands of liters. While the separation techniques used for protein purification are largely conserved across scales, the separation techniques for primary cell culture clarification vary with scale. Process models were developed to compare monoclonal antibody production costs using two cell culture clarification technologies. One process model was created for cell culture clarification by disc stack centrifugation with depth filtration. A second process model was created for clarification by multi-stage depth filtration. Analyses were performed to examine the influence of bioreactor volume, product titer, depth filter capacity, and facility utilization on overall operating costs. At bioreactor volumes <1,000 L, clarification using multi-stage depth filtration offers cost savings compared to clarification using centrifugation. For bioreactor volumes >5,000 L, clarification using centrifugation followed by depth filtration offers significant cost savings. For bioreactor volumes of ∼ 2,000 L, clarification costs are similar between depth filtration and centrifugation. At this scale, factors including facility utilization, available capital, ease of process development, implementation timelines, and process performance characterization play an important role in clarification technology selection. In the case study presented, a multi-product facility selected multi-stage depth filtration for cell culture clarification at the 500 and 2,000 L scales of operation. Facility implementation timelines, process development activities, equipment commissioning and validation, scale-up effects, and process robustness are examined.

  8. Effects of methylmercury on primary cultured rat hepatocytes: Cell injury and inhibition of growth factor stimulated DNA synthesis

    SciTech Connect

    Tanno, Keiichi; Fukazawa, Toshiyuki; Tajima, Shizuko; Fujiki, Motoo )

    1992-08-01

    Many more studies deal with the toxicity of methylmercury on nervous tissue than on its toxicity to the liver. Methylmercury accumulates in the liver in higher concentrations than brain and the liver has the primary function of detoxifying methylmercury. According to recent studies, hepatocyte mitochondrial membranes are destroyed by methylmercury and DNA synthesis is inhibited by methylmercury during hepatocyte regeneration. Methylmercury alters the membrane ion permeability of isolate skate hepatocytes, and inhibits the metal-sensitive alcohol dehydrogenase and glutathione reductase of primary cultured rat hepatocytes. However, little is known about the effect of methylmercury on hepatocyte proliferation in primary cultured rat hepatocytes. We therefore used the primary cultured rat hepatocytes to investigate the effects of methylmercury on cell injury and growth factor stimulate DNA synthesis. The primary effect of methylmercury is to inhibit hepatocyte proliferation rather than to cause direct cell injury. 16 refs., 4 figs.

  9. Between orientalism and normalization: cross-cultural lessons from Japan for a critical history of psychology.

    PubMed

    Burman, Erica

    2007-05-01

    Cross-cultural research performs a vital role within the confirmation of psychological "truths." Its differentiations work simultaneously to establish their general applicability and the superiority of Anglo-U.S. ways of living and relating. Taking three examples of how "Japan" figures within English language psychological accounts (i.e., group/individual, shame/guilt societies, and attachment styles), I indicate how the apparent stability of these truths suppressed the violent history of their generation. Moreover, I suggest how resisting the assimilation of cultural specificity into a discourse of mere variation can challenge the hegemony of Anglo-U.S. psychology and reframe the vexed question of specificity versus universality.

  10. Tissue Factor Activity in Lymphocyte Cultures from Normal Individuals and Patients with Hemophilia A

    PubMed Central

    Rickles, Frederick R.; Hardin, John A.; Pitlick, Frances A.; Hoyer, Leon W.; Conrad, Marcel E.

    1973-01-01

    The procoagulant material of lymphocytes has been characterized as tissue factor. Lymphocytes stimulated with phytohemagglutinin or the purified protein derivative of the tubercle bacillus developed procoagulant activity with incubation in tissue culture. While this material corrected the prolonged clotting time of factor VIII (AHF) deficient plasma, we have shown, utilizing a sensitive radioimmunoassay, that no AHF antigen was present in the cell cultures. Further, we have demonstrated this material to be tissue factor by coagulation techniques and immunological cross-reactivity. The published data regarding factor VIII synthesis is reviewed in light of these observations and comments are made regarding the role of the lymphocyte procoagulant. PMID:4634046

  11. Cytotoxicity evaluation using cryopreserved primary human hepatocytes in various culture formats.

    PubMed

    Richert, Lysiane; Baze, Audrey; Parmentier, Céline; Gerets, Helga H J; Sison-Young, Rowena; Dorau, Martina; Lovatt, Cerys; Czich, Andreas; Goldring, Christopher; Park, B Kevin; Juhila, Satu; Foster, Alison J; Williams, Dominic P

    2016-09-06

    Sixteen training compounds selected in the IMI MIP-DILI consortium, 12 drug-induced liver injury (DILI) positive compounds and 4 non-DILI compounds, were assessed in cryopreserved primary human hepatocytes. When a ten-fold safety margin threshold was applied, the non-DILI-compounds were correctly identified 2h following a single exposure to pooled human hepatocytes (n=13 donors) in suspension and 14-days following repeat dose exposure (3 treatments) to an established 3D-microtissue co-culture (3D-MT co-culture, n=1 donor) consisting of human hepatocytes co-cultured with non-parenchymal cells (NPC). In contrast, only 5/12 DILI-compounds were correctly identified 2h following a single exposure to pooled human hepatocytes in suspension. Exposure of the 2D-sandwich culture human hepatocyte monocultures (2D-sw) for 3days resulted in the correct identification of 11/12 DILI-positive compounds, whereas exposure of the human 3D-MT co-cultures for 14days resulted in identification of 9/12 DILI-compounds; in addition to ximelagatran (also not identified by 2D-sw monocultures, Sison-Young et al., 2016), the 3D-MT co-cultures failed to detect amiodarone and bosentan. The sensitivity of the 2D human hepatocytes co-cultured with NPC to ximelagatran was increased in the presence of lipopolysaccharide (LPS), but only at high concentrations, therefore preventing its classification as a DILI positive compound. In conclusion (1) despite suspension human hepatocytes having the greatest metabolic capacity in the short term, they are the least predictive of clinical DILI across the MIP-DILI test compounds, (2) longer exposure periods than 72h of human hepatocytes do not allow to increase DILI-prediction rate, (3) co-cultures of human hepatocytes with NPC, in the presence of LPS during the 72h exposure period allow the assessment of innate immune system involvement of a given drug.

  12. Cultural Reciprocity in Sociocultural Perspective: Adapting the Normalization Principle for Family Collaboration.

    ERIC Educational Resources Information Center

    Harry, Beth; Rueda, Robert; Kalyanpur, Maya

    1999-01-01

    Findings from a collaborative action research project involving seven culturally diverse families with children with disabilities are used to illustrate how professionals can provide assistance in a family's zone of proximal development, rather than targeting goals that are normative for the mainstream, but not for the family. (Author/CR)

  13. The Influence of Cultural Bias on Motivation to Learn English: The Case of Khoe Primary School Students in Eastern Botswana

    ERIC Educational Resources Information Center

    Magogwe, Joel Mokuedi

    2009-01-01

    This study investigated the influence of cultural bias in the teaching of English and in the books used to teach English in primary schools attended by Khoe students in eastern Botswana. The study also explored the link between cultural bias and the attitudes and motivation of Khoe students learning English. One hundred and thirty-seven students…

  14. Chinese Cultural Education in Post-Colonial Hong Kong: Primary School Chinese Language Teachers' Belief and Practice

    ERIC Educational Resources Information Center

    Kwan, Ming Kai Marko

    2010-01-01

    Before 1997, no formal curriculum on Chinese cultural education for primary schools was developed in Hong Kong although the education authority had started to introduce some items of Chinese cultural learning into the Chinese language syllabus when the Target Oriented Curriculum was implemented in 1996. However, such items were incorporated into…

  15. Xenobiotic-Metabolizing Enzyme and Transporter Gene Expression in Primary Cultures of Human Hepatocytes Modulated by Toxcast Chemicals

    EPA Science Inventory

    Primary human hepatocyte cultures are useful in vitro model systems of human liver because when cultured under appropriate conditions the hepatocytes retain liver-like functionality such as metabolism, transport, and cell signaling. This model system was used to characterize the ...

  16. Mitochondrial activity assessed by cytofluorescence after in-vitro-irradiation of primary rat brain cultures

    SciTech Connect

    Cervos-Navarro, J.; Hamdorf, G. )

    1993-05-01

    Mitochondria play a key role in cell homeostasis and are the first cell organells affected by ionizing irradiation, as it was proved by previous electron microscopic investigations. In order to observe functional parameters of mitochondria after low-dose irradiation, primary rat brain cultures (prepared from 15-day-old rat fetuses) were irradiated from a [sup 60]Co-source with 0.5 and 1 Gy at the age of 2 or 7 days in vitro (div). Cytofluorescence measurement was made by a Cytofluor[sup [trademark]2350] using Rhodamine 123. This fluorescent dye is positively charged and accumulates specifically in the mitochondria of living cells without cytotoxic effect. Since its retention depends on the negative membrane potential as well as the proton gradient that exists across the inner mitochondrial membrane, Rhodamine 123 accumulation reflects the status of mitochondrial activity as a whole. After irradiation with 0.5 and 1 Gy on day 2 in culture there was a decrease in Rhodamine uptake in the irradiated cultures during the first week after the irradiation insult which reached minimum values after 3 days. Rhodamine uptake increased during the following period and finally reached the values of the control cultures. In the second experiment with irradiated cultures on day 7 and the same doses of 0.5 and 1 Gy the accumulation of Rhodamine decreased only initially then increased tremendously. After both doses values of Rhodamine-accumulation were higher than the control level. The results demonstrated that irradiation caused a change in mitochondrial activity depending on the time of irradiation. The dramatic increase over the control levels after irradiation on day 7 in vitro is attributed to the fact that at this time synapses have already developed. Deficiency of mitochondrial activity as well as hyperactivity and the consequent change in energy production may lead to changes in neuronal metabolism including an increase in production of free radicals.

  17. Evaluation of cytokine gene expression after avian influenza virus infection in avian cell lines and primary cell cultures

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The innate immune responses elicited by avian influenza virus (AIV) infection has been studied by measuring cytokine gene expression by relative real time PCR (rRT-PCR) in vitro, using both cell lines and primary cell cultures. Continuous cell lines offer advantages over the use of primary cell cult...

  18. Using Photobleaching to Measure Spindle Microtubule Dynamics in Primary Cultures of Dividing Drosophila Meiotic Spermatocytes

    PubMed Central

    2015-01-01

    In dividing animal cells, a microtubule (MT)-based bipolar spindle governs chromosome movement. Current models propose that the spindle facilitates and/or generates translocating forces by regionally depolymerizing the kinetochore fibers (k-fibers) that bind each chromosome. It is unclear how conserved these sites and the resultant chromosome-moving mechanisms are between different dividing cell types because of the technical challenges of quantitatively studying MTs in many specimens. In particular, our knowledge of MT kinetics during the sperm-producing male meiotic divisions remains in its infancy. In this study, I use an easy-to-implement photobleaching-based assay for measuring spindle MT dynamics in primary cultures of meiotic spermatocytes isolated from the fruit fly Drosophila melanogaster. By use of standard scanning confocal microscopy features, fiducial marks were photobleached on fluorescent protein (FP)-tagged MTs. These were followed by time-lapse imaging during different division stages, and their displacement rates were calculated using public domain software. I find that k-fibers continually shorten at their poles during metaphase and anaphase A through the process of MT flux. Anaphase chromosome movement is complemented by Pac-Man, the shortening of the k-fiber at its chromosomal interface. Thus, Drosophila spermatocytes share the sites of spindle dynamism and mechanisms of chromosome movement with mitotic cells. The data reveal the applicability of the photobleaching assay for measuring MT dynamics in primary cultures. This approach can be readily applied to other systems. PMID:25802491

  19. Replication of human immunodeficiency virus type 1 in primary dendritic cell cultures.

    PubMed Central

    Langhoff, E; Terwilliger, E F; Bos, H J; Kalland, K H; Poznansky, M C; Bacon, O M; Haseltine, W A

    1991-01-01

    The ability of the human immunodeficiency virus type 1 (HIV-1) to replicate in primary blood dendritic cells was investigated. Dendritic cells compose less than 1% of the circulating leukocytes and are nondividing cells. Highly purified preparations of dendritic cells were obtained using recent advances in cell fractionation. The results of these experiments show that dendritic cells, in contrast to monocytes and T cells, support the active replication of all strains of HIV-1 tested, including T-cell tropic and monocyte/macrophage tropic isolates. The dendritic cell cultures supported much more virus production than did cultures of primary unseparated T cells, CD4+ T cells, and adherent as well as nonadherent monocytes. Replication of HIV-1 in dendritic cells produces no noticeable cytopathic effect nor does it decrease total cell number. The ability of the nonreplicating dendritic cells to support high levels of replication of HIV-1 suggests that this antigen-presenting cell population, which is also capable of supporting clonal T-cell growth, may play a central role in HIV pathogenesis, serving as a source of continued infection of CD4+ T cells and as a reservoir of virus infection. Images PMID:1910172

  20. Exposure to 1800 MHz radiofrequency radiation induces oxidative damage to mitochondrial DNA in primary cultured neurons.

    PubMed

    Xu, Shangcheng; Zhou, Zhou; Zhang, Lei; Yu, Zhengping; Zhang, Wei; Wang, Yuan; Wang, Xubu; Li, Maoquan; Chen, Yang; Chen, Chunhai; He, Mindi; Zhang, Guangbin; Zhong, Min

    2010-01-22

    Increasing evidence indicates that oxidative stress may be involved in the adverse effects of radiofrequency (RF) radiation on the brain. Because mitochondrial DNA (mtDNA) defects are closely associated with various nervous system diseases and mtDNA is particularly susceptible to oxidative stress, the purpose of this study was to determine whether radiofrequency radiation can cause oxidative damage to mtDNA. In this study, we exposed primary cultured cortical neurons to pulsed RF electromagnetic fields at a frequency of 1800 MHz modulated by 217 Hz at an average special absorption rate (SAR) of 2 W/kg. At 24 h after exposure, we found that RF radiation induced a significant increase in the levels of 8-hydroxyguanine (8-OHdG), a common biomarker of DNA oxidative damage, in the mitochondria of neurons. Concomitant with this finding, the copy number of mtDNA and the levels of mitochondrial RNA (mtRNA) transcripts showed an obvious reduction after RF exposure. Each of these mtDNA disturbances could be reversed by pretreatment with melatonin, which is known to be an efficient antioxidant in the brain. Together, these results suggested that 1800 MHz RF radiation could cause oxidative damage to mtDNA in primary cultured neurons. Oxidative damage to mtDNA may account for the neurotoxicity of RF radiation in the brain.

  1. Using Photobleaching to Measure Spindle Microtubule Dynamics in Primary Cultures of Dividing Drosophila Meiotic Spermatocytes.

    PubMed

    Savoian, Matthew S

    2015-07-01

    In dividing animal cells, a microtubule (MT)-based bipolar spindle governs chromosome movement. Current models propose that the spindle facilitates and/or generates translocating forces by regionally depolymerizing the kinetochore fibers (k-fibers) that bind each chromosome. It is unclear how conserved these sites and the resultant chromosome-moving mechanisms are between different dividing cell types because of the technical challenges of quantitatively studying MTs in many specimens. In particular, our knowledge of MT kinetics during the sperm-producing male meiotic divisions remains in its infancy. In this study, I use an easy-to-implement photobleaching-based assay for measuring spindle MT dynamics in primary cultures of meiotic spermatocytes isolated from the fruit fly Drosophila melanogaster. By use of standard scanning confocal microscopy features, fiducial marks were photobleached on fluorescent protein (FP)-tagged MTs. These were followed by time-lapse imaging during different division stages, and their displacement rates were calculated using public domain software. I find that k-fibers continually shorten at their poles during metaphase and anaphase A through the process of MT flux. Anaphase chromosome movement is complemented by Pac-Man, the shortening of the k-fiber at its chromosomal interface. Thus, Drosophila spermatocytes share the sites of spindle dynamism and mechanisms of chromosome movement with mitotic cells. The data reveal the applicability of the photobleaching assay for measuring MT dynamics in primary cultures. This approach can be readily applied to other systems.

  2. Pyrrolizidine alkaloid clivorine induced oxidative injury on primary cultured rat hepatocytes.

    PubMed

    Ji, LiLi; Liu, TianYu; Wang, ZhengTao

    2010-04-01

    Clivorine is an otonecine-type hepatotoxic pyrrolizidine alkaloid (HPAs), to which humans are exposed when consuming herbs containing such components. In the present study, we investigated clivorine-induced oxidative stress injury on primary cultured rat hepatocytes. Rat hepatocytes were treated with various concentrations of clivorine (1-100 microM) for 48 hours, and then cell viability was detected by 3-(4,5-dimethyl-thiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT) assay, while lipid peroxidation (LPO) level, glutathione peroxidase (GPx), glutathione-S-transferase (GST), glutathione reductase (GR), catalase (CAT) and superoxide dismutase (SOD) activities were determined to evaluate the oxidative injury. The results of MTT assay showed that clivorine decreased cell viability in a concentration-dependent manner. Clivorine also increased LPO amounts in rat hepatocytes at the concentrations of 50 microM and 100 microM. Further results showed that clivorine decreased GPx, GST and GR activities, which are all reduced glutathione (GSH)-related antioxidant enzymes. CAT and SOD are both important antioxidant enzymes, and the results showed that clivorine increased CAT activity at the low concentration of 5 muM and decreased cellular SOD activity at all concentrations. Taken together, our results demonstrated that clivorine induced toxicity on primary cultured rat hepatocytes by causing the damage on cellular redox balance.

  3. Fluorescence-based co-culture of normal and cancerous cells as an indicator of therapeutic effects in cancer.

    PubMed

    Tamura, Masato; Matsui, Hirofumi; Hyodo, Ichinosuke; Tanaka, Junko; Miwa, Yoshihiro

    2014-10-15

    Comprehensive evaluation of the effects of cancer therapies in vitro is difficult because of the need to distinguish the main effects from the side effects within the data. This problem cannot be overcome by methods involving monoculture, because the effects of anti-cancer drugs in a monoculture can only be measured on either normal or cancerous cells in isolation. In order to promote therapeutic development, therefore, we need a novel drug evaluation method which can simultaneously determine both therapeutic activity and toxicity under a co-culture of normal and cancerous cells. Co-culture creates a more biomimetic condition in comparison to monoculture. The novel method proposed in this study uses an easy experiment for estimating the effects of treatments with various kinds of drugs as a solution to the abovementioned problems. We have previously established two cell lines: a rat gastric mucosal cell line (RGM) and its corresponding cancerous mutant cell line (RGK). In this study, we have developed a new evaluation procedure using a co-culture of green fluorescent protein-expressing RGM cells (RGM-GFP) and kusabira orange-expressing RGK cells (RGK-KO). These cell lines emit green and red fluorescence, respectively. We demonstrated the capability of the method in evaluations of the cancer-selective effects of anti-cancer drugs and X-ray treatment. These results clearly distinguished the cancer-selective toxicity of the applied therapies.

  4. Fibronectin is not Present in the Focal Adhesions Formed between Normal Cultured Fibroblasts and Their Substrata

    NASA Astrophysics Data System (ADS)

    Chen, Wen-Tien; Singer, S. J.

    1980-12-01

    Fibronectin is an extracellular matrix protein that has been implicated in the spreading and adhesion of cultured fibroblasts to their substrata. In this paper, double immunoelectron microscopic labeling experiments for fibronectin and for concanavalin A-binding proteins on the cell surface were carried out on ultrathin frozen sections of cultures of embryonic chicken heart fibroblasts. On cross sections through the focal adhesions of the cell to the substratum there was substantial labeling for concanavalin A-binding proteins but no detectable labeling for fibronectin, whereas both the binding proteins and fibronectin were extensively labeled elsewhere on the cell surface and substratum. These results demonstrate that fibronectin is not present within the sites of focal adhesions. Therefore, the functions of fibronectin in cell spreading and adhesion are not directly mediated through its binding at focal adhesion sites. An alternative model is presented which can account for such fibronectin functions.

  5. Evaluation of MENT on primary cell cultures from benign prostatic hyperplasia and prostate carcinoma.

    PubMed

    Mendoza, Patricia; Sánchez, Catherine; Contreras, Héctor R; Vergara, Jorge; Acevedo, Cristian; Cabezas, Juan; Huidobro, Christian; Noé, Gabriela; Castellón, Enrique A

    2009-12-01

    7-alpha-Methyl-19-Nortestosterone (MENT) is a synthetic androgen more potent than testosterone (T) and cannot be reduced at 5-alpha position. No important effects of MENT on prostate growth have been reported. However, little is known about the effect of MENT on benign prostatic hyperplasia (BPH) or prostate carcinoma (CaP). We evaluate the effect of MENT, T and dihydrotestosterone (DHT) on secretion, proliferation and gene expression of primary cell cultures from human BPH and CaP. Moreover, the effect of these androgens was examined in the presence of finasteride to determine the influence of the 5-alpha reductase (5-AR) activity on the androgenic potency. BPH and CaP primary cultures were treated with 0, 1, 10 and 100 nM of T, MENT or DHT during 24 and 48 h. Prostate-specific antigen (PSA) was measured by micro particles immunoassay and proliferation rate by spectrophotometric assay (MTT) and by the immunochemical detection of the proliferation marker Ki-67. Gene expression of FGF8b (androgen sensitive gene) was evaluated by semi-quantitative RT-PCR. Results showed that MENT treatments increased PSA secretion and proliferation rate with a potency ranged between T and DHT. Similar effects of MENT were observed in both BPH and CaP cultures. The studies with finasteride showed that in BPH and CaP cells, the conversion of T into DHT significantly contributes to its effect on the proliferation and PSA secretion, and corroborated the resistance of MENT to the 5-AR. The effect of MENT on the gene expression of FGF8b in CaP cells was similar to T and lower than DHT. It is concluded that MENT increases proliferative and secretory activities and gene expression on pathological prostate cells although in less extent than the active metabolite DHT. Furthermore, the fall of endogenous concentration of T during MENT treatment anticipates that this androgen will be of low impact for the prostate.

  6. What pediatricians should know about normal language development: ensuring cultural differences are not diagnosed as disorders.

    PubMed

    Weiss, Amy L; Van Haren, Melissa S

    2003-07-01

    The roles and responsibilities of speech-language pathologists and pediatricians have become greater with the changing population demographics in the United States. In some states, the majority of the population belongs to a national cultural minority, eg, New Mexico. Even a state such as Iowa, with only a 5% nonmajority population, has a school-aged population that is almost 10% nonmajority. This growth of diversity is likely to continue. Rather than viewing sensitivity to the influence of culture on language learning and other developmental areas as an "add-on" to a practice, it may be wiser to recognize that approaching all clients with as few assumptions about their behaviors as possible will guarantee nonbiased service delivery for all. Without nonbiased service delivery, incorrect diagnoses and provision of inappropriate therapy become more likely. Fortunately, many resources are available to assist pediatricians and speech-language pathologists in learning about various cultures. Institutional review boards have become more vigilant about the inclusion of a cross-section of subject populations as participants in research studies in addition to protecting the rights of all participants. Funding agencies also have expressed as a priority the inclusion of research subjects from minority populations to add to the information available about the incidence and prevalence of disorders across the range of our potential patients. In a society in which cultural differences are not just defined by race or ethnicity, but by gender, sexual orientation, age, geographic region, and religion, belief systems about disease, disability, and treatment are dynamic entities for health professionals to take into consideration. It is a challenge that speech-language pathologists and pediatricians must meet if they are to provide the best and most appropriate services for their patients.

  7. Expression of adipocyte biomarkers in a primary cell culture models reflects preweaning adipobiology.

    PubMed

    Chu, Dinh-Toi; Malinowska, Elzbieta; Gawronska-Kozak, Barbara; Kozak, Leslie P

    2014-06-27

    A cohort of genes was selected to characterize the adipogenic phenotype in primary cell cultures from three tissue sources. We compared the quantitative expression of biomarkers in culture relative to their expression in vivo because the mere presence or absence of expression is minimally informative. Although all biomarkers analyzed have biochemical functions in adipocytes, the expression of some of the biomarkers varied enormously in culture relative to their expression in the adult fat tissues in vivo, i.e. inguinal fat for white adipocytes and brite cells, interscapular brown adipose tissue for brown adipocytes, and ear mesenchymal stem cells for white adipocytes from adult mice. We propose that the pattern of expression in vitro does not reflect gene expression in the adult mouse; rather it is predominantly the expression pattern of adipose tissue of the developing mouse between birth and weaning. The variation in gene expression among fat depots in both human and rodent has been an extensively studied phenomenon, and as recently reviewed, it is related to subphenotypes associated with immune function, the inflammatory response, fat depot blood flow, and insulin sensitivity. We suggest that adipose tissue biology in the period from birth to weaning is not just a staging platform for the emergence of adult white fat but that it has properties to serve the unique needs of energy metabolism in the newborn. A case in point is the differentiation of brite cells that occurs during this period followed by their involution immediately following weaning.

  8. Establishment, characterization, and toxicological application of loggerhead sea turtle (Caretta caretta) primary skin fibroblast cell cultures.

    PubMed

    Webb, Sarah J; Zychowski, Gregory V; Bauman, Sandy W; Higgins, Benjamin M; Raudsepp, Terje; Gollahon, Lauren S; Wooten, Kimberly J; Cole, Jennifer M; Godard-Codding, Céline

    2014-12-16

    Pollution is a well-known threat to sea turtles but its impact is poorly understood. In vitro toxicity testing presents a promising avenue to assess and monitor the effects of environmental pollutants in these animals within the legal constraints of their endangered status. Reptilian cell cultures are rare and, in sea turtles, largely derived from animals affected by tumors. Here we describe the full characterization of primary skin fibroblast cell cultures derived from biopsies of multiple healthy loggerhead sea turtles (Caretta caretta), and the subsequent optimization of traditional in vitro toxicity assays to reptilian cells. Characterization included validating fibroblast cells by morphology and immunocytochemistry, and optimizing culture conditions by use of growth curve assays with a fractional factorial experimental design. Two cell viability assays, MTT and lactate dehydrogenase (LDH), and an assay measuring cytochrome P4501A (CYP1A) expression by quantitative PCR were optimized in the characterized cells. MTT and LDH assays confirmed cytotoxicity of perfluorooctanoic acid at 500 μM following 72 and 96 h exposures while CYP1A5 induction was detected after 72 h exposure to 0.1-10 μM benzo[a]pyrene. This research demonstrates the validity of in vitro toxicity testing in sea turtles and highlights the need to optimize mammalian assays to reptilian cells.

  9. U. v. -enhanced reactivation of u. v. -irradiated herpes virus by primary cultures of rat hepatocytes

    SciTech Connect

    Zurlo, J.; Yager, J.D. )

    1984-04-01

    Carcinogen treatment of cultured mammalian cells prior to infection with u.v.-irradiated virus results in enhanced virus survival and mutagenesis suggesting the induction of SOS-type processes. The development of a primary rat hepatocyte culture system is reported to investigate cellular responses to DNA damage which may be relevant to hepatocarcinogenesis in vivo. Enhanced reactivation of u.v.-irradiated Herpes simplex virus type 1 (HSV-1) occurred in hepatocytes irradiated with u.v. Cultured hepatocytes were pretreated with u.v. at the time of enhanced DNA synthesis. These treatments caused an inhibition followed by a recovery of DNA synthesis. At various times after pretreatment, the hepatocytes were infected with control or u.v.-irradiated HSV-1 at low multiplicity, and virus survival was measured. U.v.-irradiated HSV-1 exhibited the expected two-component survival curve in control or u.v. pretreated hepatocytes. The magnitude of enhanced reactivation of HSV-1 was dependent on the u.v. dose to the hepatocytes, the time of infection following u.v. pretreatment, and the level of DNA synthesis at the time of pretreatment. These results suggest that u.v. treatment of rat hepatocytes causes the induction of SOS-type functions tht may have a role in the initiation of hepatocarcinogenesis.

  10. An imbalance in progenitor cell populations reflects tumour progression in breast cancer primary culture models

    PubMed Central

    2011-01-01

    Background Many factors influence breast cancer progression, including the ability of progenitor cells to sustain or increase net tumour cell numbers. Our aim was to define whether alterations in putative progenitor populations could predict clinicopathological factors of prognostic importance for cancer progression. Methods Primary cultures were established from human breast tumour and adjacent non-tumour tissue. Putative progenitor cell populations were isolated based on co-expression or concomitant absence of the epithelial and myoepithelial markers EPCAM and CALLA respectively. Results Significant reductions in cellular senescence were observed in tumour versus non-tumour cultures, accompanied by a stepwise increase in proliferation:senescence ratios. A novel correlation between tumour aggressiveness and an imbalance of putative progenitor subpopulations was also observed. Specifically, an increased double-negative (DN) to double-positive (DP) ratio distinguished aggressive tumours of high grade, estrogen receptor-negativity or HER2-positivity. The DN:DP ratio was also higher in malignant MDA-MB-231 cells relative to non-tumourogenic MCF-10A cells. Ultrastructural analysis of the DN subpopulation in an invasive tumour culture revealed enrichment in lipofuscin bodies, markers of ageing or senescent cells. Conclusions Our results suggest that an imbalance in tumour progenitor subpopulations imbalances the functional relationship between proliferation and senescence, creating a microenvironment favouring tumour progression. PMID:21521500

  11. Activity of Protease-Activated Receptors in Primary Cultured Human Myenteric Neurons

    PubMed Central

    Kugler, Eva M.; Mazzuoli, Gemma; Demir, Ihsan E.; Ceyhan, Güralp O.; Zeller, Florian; Schemann, Michael

    2012-01-01

    Activity of the four known protease-activated receptors (PARs) has been well studied in rodent enteric nervous system and results in animal models established an important role for neuronal PAR2. We recently demonstrated that, unlike in rodents, PAR1 is the dominant neuronal protease receptor in the human submucous plexus. With this study we investigated whether this also applies to the human myenteric plexus. We used voltage sensitive dye recordings to detect action potential discharge in primary cultures of human myenteric neurons in response to PAR activating peptides (APs). Application of the PAR1-AP (TFLLR) or PAR4-AP (GYPGQV) evoked spike discharge in 79 or 23% of myenteric neurons, respectively. The PAR1-AP response was mimicked by the endogenous PAR1 activator thrombin and blocked by the PAR1 antagonists SCH79797. Human myenteric neurons did not respond to PAR2-AP. This was not due to culture conditions because all three PAR-APs evoked action potentials in cultured guinea pig myenteric neurons. Consecutive application of PAR-APs revealed coexpression (relative to the population responding to PAR-APs) of PAR1/PAR2 in 51%, PAR1/PAR4 in 43%, and of PAR2/PAR4 in 29% of guinea pig myenteric neurons. Our study provided further evidence for the prominent role of neuronal PAR1 in the human enteric nervous system. PMID:22988431

  12. In vitro culture of oocytes and granulosa cells collected from normal, obese, emaciated and metabolically stressed ewes.

    PubMed

    Tripathi, S K; Farman, M; Nandi, S; Mondal, S; Gupta, Psp; Kumar, V Girish

    2016-07-01

    The present study was undertaken to investigate the oocyte morphology, its fertilizing capacity and granulosa cell functions in ewes (obese, normal, metabolic stressed and emaciated). Ewes (Ovis aries) of approximately 3 years of age (Bellary breed) from a local village were screened, chosen and categorized into a) normal b) obese but not metabolically stressed, c) Emaciated but not metabolically stressed d) Metabolically stressed based on body condition scoring and blood markers. Oocytes and granulosa cells were collected from ovaries of the ewes of all categories after slaughter and were classified into good (oocytes with more than three layers of cumulus cells and homogenous ooplasm), fair (oocytes one or two layers of cumulus cells and homogenous ooplasm) and poor (denuded oocytes or with dark ooplasm). The good and fair quality oocytes were in vitro matured and cultured with fresh semen present and the fertilization, cleavage and blastocyst development were observed. The granulosa cells were cultured for evaluation of metabolic activity by use of the MTT assay, and cell viability, cell number as well as estrogen and progesterone production were assessed. It was observed that the good and fair quality oocytes had greater metabolic activity when collected from normal and obese ewes compared with those from emaciated and metabolically stressed ewes. No significant difference was observed in oocyte quality and maturation amongst the oocytes collected from normal and obese ewes. The cleavage and blastocyst production rates were different for the various body condition classifications and when ranked were: normal>obese>metabolically stressed>emaciated. Lesser metabolic activity was observed in granulosa cells obtained from ovaries of emaciated ewes. However, no changes were observed in viability and cell number of granulosa cells obtained from ewes with the different body condition categories. Estrogen and progesterone production from cultured granulosa cells were

  13. Wnt3a induces exosome secretion from primary cultured rat microglia

    PubMed Central

    2012-01-01

    Background Microglia, the immune effector cells of the CNS and the signaling molecule Wnt, both play critical roles in neurodevelopment and neurological disease. Here we describe the inducible release of exosomes from primary cultured rat microglia following treatment with recombinant carrier-free Wnt3a. Results Wnt3a was internalised into microglia, being detectable in early endosomes, and secreted in exosomes through a GSK3-independent mechanism. Electron microscopy demonstrated that exosomes were elliptical, electron-dense (100 nm) vesicles that coalesced with time in vitro. In contrast to microglia, primary cortical neurons released exosomes constitutively and the quantity of exosomes released was not altered by Wnt3a treatment. The proteomic profile of the microglial-derived exosomes was characterised using liquid chromatography-tandem mass spectrometry (LC/MS/MS) and the vesicles were found to be associated with proteins involved in cellular architecture, metabolism, protein synthesis and protein degradation including β-actin, glyceraldehyde-3-phosphate dehydrogenase, ribosomal subunits and ubiquitin (45 proteins in total). Unlike lipopolysaccharide, Wnt3a did not induce a neurotoxic, pro-inflammatory phenotype in primary microglia. Conclusion These findings reveal a novel mechanism through which Wnt3a signals in microglia resulting in the release of exosomes loaded with proteinaceous cargo. PMID:23173708

  14. Nontargeted Stressful Effects in Normal Human Fibroblast Cultures Exposed to Low Fluences of High Charge, High Energy (HZE) Particles: Kinetics of Biologic Responses and Significance of Secondary Radiations

    PubMed Central

    Gonon, Géraldine; Groetz, Jean-Emmanuel; de Toledo, Sonia M.; Howell, Roger W.; Fromm, Michel; Azzam, Edouard I.

    2014-01-01

    The induction of nontargeted stressful effects in cell populations exposed to low fluences of high charge (Z) and high energy (E) particles is relevant to estimates of the health risks of space radiation. We investigated the up-regulation of stress markers in confluent normal human fibroblast cultures exposed to 1,000 MeV/u iron ions [linear energy transfer (LET) ~151 keV/μm] or 600 MeV/u silicon ions (LET ~50 keV/μm) at mean absorbed doses as low as 0.2 cGy, wherein 1–3% of the cells were targeted through the nucleus by a primary particle. Within 24 h postirradiation, significant increases in the levels of phospho-TP53 (serine 15), p21Waf1 (CDKN1A), HDM2, phospho-ERK1/2, protein carbonylation and lipid peroxidation were detected, which suggested participation in the stress response of cells not targeted by primary particles. This was supported by in situ studies that indicated greater increases in 53BP1 foci formation, a marker of DNA damage. than expected from the number of primary particle traversals. The effect was expressed as early as 15 min after exposure, peaked at 1 h and decreased by 24 h. A similar tendency occurred after exposure of the cell cultures to 0.2 cGy of 3.7 MeV α particles (LET ~109 keV/μm) that targets ~1.6% of nuclei, but not after 0.2 cGy from 290 MeV/u carbon ions (LET ~13 keV/μm) by which, on average, ~13% of the nuclei were hit, which highlights the importance of radiation quality in the induced effect. Simulations with the FLUKA multi-particle transport code revealed that fragmentation products, other than electrons, in cell cultures exposed to HZE particles comprise <1% of the absorbed dose. Further, the radial spread of dose due to secondary heavy ion fragments is confined to approximately 10–20 μm. Thus, the latter are unlikely to significantly contribute to stressful effects in cells not targeted by primary HZE particles. PMID:23465079

  15. Development of primary cell cultures using hemocytes and phagocytic tissue cells of Locusta migratoria: an application for locust immunity studies.

    PubMed

    Duressa, Tewodros Firdissa; Huybrechts, Roger

    2016-01-01

    Insect cell cultures played central roles in unraveling many insect physiological and immunological processes. Regardless, despite imminent needs, insect cell lines were developed primarily from Dipteran and Lepidopteran orders, leaving many important insects such as Orthopteran locusts under-represented. Besides the lack of cell lines, the slow progress in development of in vitro techniques is attributed to poor communications between different laboratories regarding optimized primary cell cultures. Therefore, we report here about methods developed for primary cell culture of Locusta migratoria hemocyte and phagocytic tissue cells by which we could maintain viable hemocytes in vitro for over 5 d and phagocytic tissue cells for over 12 d. 2-Mercaptoethanol and phenyl-thiourea supplements in Grace's medium together with addition of fetal bovine serum 30 min after cell seeding resulted in a successful setup of the primary cell cultures and a week-long survival of the hemocytes and phagocytic tissue cells in vitro.

  16. Primary transitions between the yrast superdeformed band and low-lying normal deformed states in {sup 194}Pb

    SciTech Connect

    Hauschild, K.; Bernstein, L.A.; Becker, J.A.

    1996-12-31

    The observation of one-step `primary` gamma-ray transitions directly linking the superdeformed (SD) states to the normal deformed (ND) low-lying states of known excitation energies (E{sub x}), spins and parities (J{sup {pi}}) is crucial to determining the E{sub x} and J{sup {pi}} of the SD states. With this knowledge one can begin to address some of the outstanding problems associated with SD nuclei, such as the identical band issue, and one can also place more stringent restrictions on theoretical calculations which predict SD states and their properties. Brinkman, et al., used the early implementation of the GAMMASPHERE spectrometer array (32 detectors) and proposed a single, candidate {gamma} ray linking the {sup 194}Pb yrast SD band to the low-lying ND states in {sup 194}Pb. Using 55 detectors in the GAMMASPHERE array Khoo, et al., observed multiple links between the yrast SD band in {sup 194}Hg and the low-lying level scheme and conclusively determined E{sub x} and J of the yrast SD states. Here the authors report on an experiment in which Gammasphere with 88 detectors was used and the E{sub x} and J{sup {pi}} values of the yrast SD states in {sup 194}Pb were uniquely determined. Twelve one-step linking transitions between the yrast SD band and low-lying states in {sup 194}Pb have been identified, including the transition proposed by Brinkman. These transitions have been placed in the level scheme of {sup 194}Pb using coincidence relationships and agreements between the energies of the primary transitions and the energy differences in level spacings. Furthermore, measurements of angular asymmetries have yielded the multipolarities of the primaries which have allowed J{sup {pi}} assignments of the {sup 194}Pb SD states to be unambiguously determined for the first time without a priori assumptions about the character of SD bands. A study performed in parallel to this work using the EUROGAM-II array reports similar, but somewhat less extensive, results.

  17. Mechanisms of chloroform and carbon tetrachloride toxicity in primary cultured mouse hepatocytes.

    PubMed Central

    Ruch, R J; Klaunig, J E; Schultz, N E; Askari, A B; Lacher, D A; Pereira, M A; Goldblatt, P J

    1986-01-01

    Mechanisms of chloroform (CHCl3) and carbon tetrachloride (CCl4) toxicity to primary cultured male B6C3F1 mouse hepatocytes were investigated. The cytotoxicity of both CHCl3 and CCl4 was dose- and duration-dependent. Maximal hepatocyte toxicity, as determined by lactate dehydrogenase leakage into the culture medium, occurred with the highest concentrations of CHCl3 (5 mM) and CCl4 (2.5 mM) used and with the longest duration of treatment (20 hr). CCl4 was approximately 16 times more toxic than CHCl3 to the hepatocytes. The toxicity of these compounds was decreased by adding the mixed function oxidase system (MFOS) inhibitor, SKF-525A (25 microM) to the cultures. The addition of diethyl maleate (0.25 mM), which depletes intracellular glutathione (GSH)-potentiated CHCl3 and CCl4 toxicity. The toxicity of CHCl3 and CCl4 could also be decreased by adding the antioxidants N,N'-diphenyl-p-phenylenediamine (DPPD) (25 microM), alpha-tocopherol acetate (Vitamin E) (0.1 mM), or superoxide dismutase (SOD) (100 U/mL) to the cultures. These results suggest that: in mouse hepatocytes, both CHCl3 and CCl4 are metabolized to toxic components by the MFOS; GSH plays a role in detoxifying those metabolites; free radicals are produced during the metabolism of CHCl3 and CCl4; and free radicals may be important mediators of the toxicity of these two halomethanes. PMID:3816733

  18. Mechanisms of chloroform and carbon tetrachloride toxicity in primary cultured mouse hepatocytes

    SciTech Connect

    Ruch, R.J.; Klaunig, J.E.; Schultz, N.E.; Askari, A.B.; Lacher, D.A.; Pereira, M.A.; Goldblatt, P.J.

    1986-11-01

    Mechanisms of chloroform (CHCl/sub 3/) and carbon tetrachloride (CCl/sub 4/) toxicity to primary cultured male B6C3F1 mouse hepatocytes were investigated. The cytotoxicity of both CHCl/sub 3/ and CCl/sub 4/ was dose- and duration-dependent. Maximal hepatocyte toxicity, as determined by lactate dehydrogenase leakage into the culture medium, occurred with the highest concentrations of CHCl/sub 3/ (5 mM) and CCl/sub 4/ (2.5 mM) used and with the longest duration of treatment (20 hr). CCl/sub 4/ was approximately 16 times more toxic than CHCl/sub 3/ to the hepatocytes. The toxicity of these compounds was decreased by adding the mixed function oxidase system (MFOS) inhibitor, SKF-525A (25..mu..M) to the cultures. The addition of diethyl maleate (0.25 mM), which depletes intracellular glutathione (GSH)-potentiated CHCl/sub 3/ and CCl/sub 4/ toxicity. The toxicity of CHCl/sub 3/ and CCl/sub 4/ could also be decreased by adding the antioxidants N,N'-diphenyl-p-phenylenediamine (DPPD) (25..mu..M), ..cap alpha..-tocopherol acetate (Vitamin E) (0.1 mM), or superoxide dismutase (SOD) (100 U/mL) to the cultures. These results suggest that: in mouse hepatocytes, both CHCl/sub 3/ and CCl/sub 4/ are metabolized to toxic components by the MFOS; GSH plays a role in detoxifying those metabolites; free radicals are produced during the metabolism of CHCl/sub 3/ and CCl/sub 4/; and free radicals may be important mediators of the toxicity of these two halomethanes.

  19. Ultrastructural characterization of normal and abnormal chondrogenesis in micromass rat embryo limb bud cell cultures.

    PubMed

    Renault, J Y; Caillaud, J M; Chevalier, J

    1995-02-01

    Inhibition of chondrogenesis in limb bud cell micromass cultures has been proposed as a short-term teratogen detection test. Validation studies were performed by testing large series of reference compounds and comparing their teratogenic potential with their ability to inhibit chondrogenesis; however, there are few reports describing the histological and ultrastructural changes associated with inhibition of chondrogenesis in vitro. The objective of this study was to provide a qualitative description of the histological and ultrastructural alterations induced by three chondrogenesis inhibitors: retinoic acid (RA) and 6-aminonicotinamide (6AN), two teratogens, and doxylamine succinate (DS), a nonteratogen compound. In addition, in order to have a basis for the interpretation of the morphological alterations induced by the test compounds, the histological and ultrastructural changes which occur during the time course of chondrogenesis in control cultures were described and compared with those in rat embryo limb buds. We found that RA at 0.5 micrograms/ml led to a marked decrease in the number and size of cartilaginous foci; most cells lacked morphological signs of differentiation but their ability to proliferate was unaffected. At concentrations of 2 micrograms/ml and more, 6AN delayed cell proliferation, reduced staining of the extracellular matrix, and induced the formation of endoplasmic cisternae. DS at 50 micrograms/ml affected both differentiation and proliferation; pigment deposits were observed in chondrocytes, suggesting phospholipid metabolism disorders. In conclusion, this study showed that inhibition of chondrogenesis in this simple cell culture system can be associated with different types of histological and ultrastructural alterations. Examination of these alterations can provide useful information about the teratogenic potential of tested compounds and their mechanism of action.

  20. Comparative Analysis of Human and Rodent Brain Primary Neuronal Culture Spontaneous Activity Using Micro-Electrode Array Technology.

    PubMed

    Napoli, Alessandro; Obeid, Iyad

    2016-03-01

    Electrical activity in embryonic brain tissue has typically been studied using Micro Electrode Array (MEA) technology to make dozens of simultaneous recordings from dissociated neuronal cultures, brain stem cell progenitors, or brain slices from fetal rodents. Although these rodent neuronal primary culture electrical properties are mostly investigated, it has not been yet established to what extent the electrical characteristics of rodent brain neuronal cultures can be generalized to those of humans. A direct comparison of spontaneous spiking activity between rodent and human primary neurons grown under the same in vitro conditions using MEA technology has never been carried out before and will be described in the present study. Human and rodent dissociated fetal brain neuronal cultures were established in-vitro by culturing on a glass grid of 60 planar microelectrodes neurons under identical conditions. Three different cultures of human neurons were produced from tissue sourced from a single aborted fetus (at 16-18 gestational weeks) and these were compared with seven different cultures of embryonic rat neurons (at 18 gestational days) originally isolated from a single rat. The results show that the human and rodent cultures behaved significantly differently. Whereas the rodent cultures demonstrated robust spontaneous activation and network activity after only 10 days, the human cultures required nearly 40 days to achieve a substantially weaker level of electrical function. These results suggest that rat neuron preparations may yield inferences that do not necessarily transfer to humans.

  1. Putative Nanobacteria Represent Physiological Remnants and Culture By-Products of Normal Calcium Homeostasis

    PubMed Central

    Young, John D.; Young, Lena; Wu, Cheng-Yeu; Young, Andrew

    2009-01-01

    Putative living entities called nanobacteria (NB) are unusual for their small sizes (50–500 nm), pleomorphic nature, and accumulation of hydroxyapatite (HAP), and have been implicated in numerous diseases involving extraskeletal calcification. By adding precipitating ions to cell culture medium containing serum, mineral nanoparticles are generated that are morphologically and chemically identical to the so-called NB. These nanoparticles are shown here to be formed of amorphous mineral complexes containing calcium as well as other ions like carbonate, which then rapidly acquire phosphate, forming HAP. The main constituent proteins of serum-derived NB are albumin, fetuin-A, and apolipoprotein A1, but their involvement appears circumstantial since so-called NB from different body fluids harbor other proteins. Accordingly, by passage through various culture media, the protein composition of these particles can be modulated. Immunoblotting experiments reveal that antibodies deemed specific for NB react in fact with either albumin, fetuin-A, or both, indicating that previous studies using these reagents may have detected these serum proteins from the same as well as different species, with human tissue nanoparticles presumably absorbing bovine serum antigens from the culture medium. Both fetal bovine serum and human serum, used earlier by other investigators as sources of NB, paradoxically inhibit the formation of these entities, and this inhibition is trypsin-sensitive, indicating a role for proteins in this inhibitory process. Fetuin-A, and to a lesser degree albumin, inhibit nanoparticle formation, an inhibition that is overcome with time, ending with formation of the so-called NB. Together, these data demonstrate that NB are most likely formed by calcium or apatite crystallization inhibitors that are somehow overwhelmed by excess calcium or calcium phosphate found in culture medium or in body fluids, thereby becoming seeds for calcification. The structures

  2. Putative nanobacteria represent physiological remnants and culture by-products of normal calcium homeostasis.

    PubMed

    Young, John D; Martel, Jan; Young, Lena; Wu, Cheng-Yeu; Young, Andrew; Young, David

    2009-01-01

    Putative living entities called nanobacteria (NB) are unusual for their small sizes (50-500 nm), pleomorphic nature, and accumulation of hydroxyapatite (HAP), and have been implicated in numerous diseases involving extraskeletal calcification. By adding precipitating ions to cell culture medium containing serum, mineral nanoparticles are generated that are morphologically and chemically identical to the so-called NB. These nanoparticles are shown here to be formed of amorphous mineral complexes containing calcium as well as other ions like carbonate, which then rapidly acquire phosphate, forming HAP. The main constituent proteins of serum-derived NB are albumin, fetuin-A, and apolipoprotein A1, but their involvement appears circumstantial since so-called NB from different body fluids harbor other proteins. Accordingly, by passage through various culture media, the protein composition of these particles can be modulated. Immunoblotting experiments reveal that antibodies deemed specific for NB react in fact with either albumin, fetuin-A, or both, indicating that previous studies using these reagents may have detected these serum proteins from the same as well as different species, with human tissue nanoparticles presumably absorbing bovine serum antigens from the culture medium. Both fetal bovine serum and human serum, used earlier by other investigators as sources of NB, paradoxically inhibit the formation of these entities, and this inhibition is trypsin-sensitive, indicating a role for proteins in this inhibitory process. Fetuin-A, and to a lesser degree albumin, inhibit nanoparticle formation, an inhibition that is overcome with time, ending with formation of the so-called NB. Together, these data demonstrate that NB are most likely formed by calcium or apatite crystallization inhibitors that are somehow overwhelmed by excess calcium or calcium phosphate found in culture medium or in body fluids, thereby becoming seeds for calcification. The structures described

  3. Elevated Plasma Endothelin-1 Levels in Normal Tension Glaucoma and Primary Open-Angle Glaucoma: A Meta-Analysis

    PubMed Central

    Li, Shengjie; Zhang, Aiping

    2016-01-01

    Purpose. The aim of this meta-analysis was to clarify the association between the plasma endothelin-1 level and the risks of normal tension glaucoma (NTG) and primary open-angle glaucoma (POAG). Methods. Relevant publications were collected from three databases including PubMed, EMBASE, and the Web of Science through December 31, 2015. In this study, the terms “(endothelin OR ET) AND glaucoma” were searched. Review Manager 5.2 was used to process the data. Results. Seven studies (212 cases, 164 controls) were included for the NTG analysis. The mean plasma endothelin-1 level in the NTG subjects was 0.60 pg/mL (p = 0.02, 95% CI: 0.17–1.04) higher than that of the healthy controls. Six studies (160 cases, 174 controls) were included for the POAG analysis, and the endothelin-1 level was 0.63 pg/mL (p = 0.007, 95% CI: 0.12–1.15) higher in the POAG subjects than in the healthy controls. Additionally, two studies influenced the meta-analysis results regarding the association of plasma endothelin-1 with POAG by sensitivity analysis, and the probability of publication bias was low. Conclusions. The observation that NTG and POAG subjects showed significantly elevated endothelin-1 plasma concentrations suggests that a higher plasma level of endothelin-1 might increase the risk of NTG and POAG development. PMID:27965889

  4. TEAD transcription factors are required for normal primary myoblast differentiation in vitro and muscle regeneration in vivo

    PubMed Central

    Joshi, Shilpy; Le Gras, Stéphanie; Watanabe, Shuichi; Braun, Thomas

    2017-01-01

    The TEAD family of transcription factors (TEAD1-4) bind the MCAT element in the regulatory elements of both growth promoting and myogenic differentiation genes. Defining TEAD transcription factor function in myogenesis has proved elusive due to overlapping expression of family members and their functional redundancy. We show that silencing of either Tead1, Tead2 or Tead4 did not effect primary myoblast (PM) differentiation, but that their simultaneous knockdown strongly impaired differentiation. In contrast, Tead1 or Tead4 silencing impaired C2C12 differentiation showing their different contributions in PMs and C2C12 cells. Chromatin immunoprecipitation identified enhancers associated with myogenic genes bound by combinations of Tead4, Myod1 or Myog. Tead4 regulated distinct gene sets in C2C12 cells and PMs involving both activation of the myogenic program and repression of growth and signaling pathways. ChIP-seq from mature mouse muscle fibres in vivo identified a set of highly transcribed muscle cell-identity genes and sites bound by Tead1 and Tead4. Although inactivation of Tead4 in mature muscle fibres caused no obvious phenotype under normal conditions, notexin-induced muscle regeneration was delayed in Tead4 mutants suggesting an important role in myogenic differentiation in vivo. By combining knockdown in cell models in vitro with Tead4 inactivation in muscle in vivo, we provide the first comprehensive description of the specific and redundant roles of Tead factors in myogenic differentiation. PMID:28178271

  5. Anisotonic media and glutamate-induced ion transport and volume responses in primary astrocyte cultures.

    PubMed

    Kimelberg, H K

    1987-01-01

    1. The responses of primary monolayer astrocyte cultures prepared from neonatal rat brains to hyper- and hypotonic media and to the addition of L-glutamic acid were examined as part of a systematic approach to use these cultures to obtain information on the mechanisms of the volume changes seen in astroglial cells in situ. 2. Addition of 200 mM mannitol to the medium to make it hypertonic caused cell shrinkage as measured with [14C]3-O-methyl-D-glucose, and also activated K+ and Cl- uptake measured with 86Rb+ and 36Cl- respectively. The increased ion uptake was completely inhibited by 0.1 mM bumetanide, showing that the Na+ + K+ + 2 Cl- co-transport system was being activated by cell shrinkage. 3. Studies of 86Rb+ uptake as a function of external K+ and hypertonic media showed a complex pattern. Increased bumetanide-sensitive, hypertonic-stimulated uptake of 86Rb+ was seen up to 20 mM K+0, with maximum stimulation being first reached at around 2 to 5 mM K+. At concentrations greater than 20 mM K+0 there was a further increase in bumetanide-sensitive 86Rb+ uptake, but there was no stimulation of this uptake by hypertonicity. There were also increases in bumetanide-insensitive 86Rb+ fluxes at [K+]0 higher than 20 mM that may have been due to opening of voltage-dependent K+ channels; this increased 86Rb+ flux was decreased in hypertonic medium. 4. When primary astrocyte cultures were swollen in hypotonic medium there was a rapid increase in volume as measured with [14C] 3-O-methyl-D-glucose, which then decreased in the continued presence of hypotonic medium. Thus, these cells exhibit volume regulatory decrease or RVD, as described for other cells. The possible ionic bases of this phenomenon have not yet been fully examined but the initial RVD did not appear to stimulate a furosemide-sensitive cotransport system. 5. Glutamate has been implicated as a possible endogenous effector of volume change in astrocytes. In the presence of ouabain, L-glutamate led to swelling of

  6. Understanding metal homeostasis in primary cultured neurons. Studies using single neuron subcellular and quantitative metallomics.

    PubMed

    Colvin, Robert A; Lai, Barry; Holmes, William R; Lee, Daewoo

    2015-07-01

    The purpose of this study was to demonstrate how single cell quantitative and subcellular metallomics inform us about both the spatial distribution and cellular mechanisms of metal buffering and homeostasis in primary cultured neurons from embryonic rat brain, which are often used as models of human disease involving metal dyshomeostasis. The present studies utilized synchrotron radiation X-ray fluorescence (SRXRF) and focused primarily on zinc and iron, two abundant metals in neurons that have been implicated in the pathogenesis of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. Total single cell contents for calcium, iron, zinc, copper, manganese, and nickel were determined. Resting steady state zinc showed a diffuse distribution in both soma and processes, best defined by the mass profile of the neuron with an enrichment in the nucleus compared with the cytoplasm. Zinc buffering and homeostasis was studied using two modes of cellular zinc loading - transporter and ionophore (pyrithione) mediated. Single neuron zinc contents were shown to statistically significantly increase by either loading method - ionophore: 160 million to 7 billion; transporter 160 million to 280 million atoms per neuronal soma. The newly acquired and buffered zinc still showed a diffuse distribution. Soma and processes have about equal abilities to take up zinc via transporter mediated pathways. Copper levels are distributed diffusely as well, but are relatively higher in the processes relative to zinc levels. Prior studies have observed iron puncta in certain cell types, but others have not. In the present study, iron puncta were characterized in several primary neuronal types. The results show that iron puncta could be found in all neuronal types studied and can account for up to 50% of the total steady state content of iron in neuronal soma. Although other metals can be present in iron puncta, they are predominantly iron containing and do not appear to be

  7. Melanogenesis and antioxidant defense system in normal human melanocytes cultured in the presence of chlorpromazine.

    PubMed

    Otreba, Michał; Wrześniok, Dorota; Beberok, Artur; Rok, Jakub; Buszman, Ewa

    2015-02-01

    Chlorpromazine is used in the treatment of schizophrenia and psychotic disorders and belongs to phenothiazine class of neuroleptic drugs. It shows severe side effects such as extrapyramidal symptoms as well as ocular and skin disorders, but the mechanism is still not fully established. The aim of this study was to examine the effect of chlorpromazine on cell viability, melanogenesis and antioxidant defense system in normal human melanocytes. It has been demonstrated that chlorpromazine induces concentration dependent loss in cell viability. The value of EC(50) was calculated to be 2.53 μM. Chlorpromazine in lower concentrations (0.0001, 0.001 and 0.01 μM) increased the melanin and microphthalmia-associated transcription factor (MITF) content and tyrosinase activity, while changes of antioxidant enzymes activity were not observed. It suggests that long-term chlorpromazine therapy, even with low drug doses, may lead to hyperpigmentation disorders in skin and/or eye. The use of the analyzed drug in higher concentrations (0.1 and 1.0 μM) caused significant alterations of antioxidant enzymes activity in normal melanocytes, what may explain a potential role of chlorpromazine in the depletion of cellular antioxidant status leading to other adverse effects associated with the high-dose and/or long-term therapy.

  8. Tissue culture of normal and cystic fibrosis sweat gland duct cells. I. Alterations in dome formation.

    PubMed

    Hazen-Martin, D J; Spicer, S S; Sens, M A; Jenkins, M Q; Westphal, M C; Sens, D A

    1987-01-01

    The elucidation of the underlying defect in fluid secretion by cystic fibrosis (CF) sweat glands is hindered by the unavailability of an experimental model for investigating this disease. As a potential model system, a serum-free growth medium was developed that supports the explant growth of epithelial cells from fragments of human skin. Immunohistochemical analysis demonstrated that these epithelial cell outgrowths originated from the duct of the sweat gland. By electron microscopy, the cells were demonstrated to possess keratinocyte-like morphology as noted by the presence of a multilayered outgrowth of cells containing well-defined keratin bundles. Identical outgrowths from skin biopsies of CF patients were compared to normal outgrowths and alterations were noted to occur in dome formation and in the number of intercellular spaces between cells. Doming alterations were also noted to occur in the CF heterozygous state. No differences in cell fine structure or in growth factor requirements for cell proliferation were noted between normal and CF cells. The potential use of this system as a model for CF research is discussed.

  9. [Mycrob-1000: an alternative for the rapid determination of urine culture in the primary health level].

    PubMed

    Alarcón, Rolando Contreras; Ruiz, Fernando Travieso; Tamayo, Angela Zayas; Carmona, Gloria Roura; Varela, Estrella Alvarez; Ochoa, Gilberto Tillán; Frómeta, Nardo Ramírez

    2004-01-01

    The use of equipment Mycrob-1000 in detecting urinary infections in 4 hours in a primary health care center is evaluated. Two hundred fifty eight urine samples obtained from spontaneous miction were processed; the reference method was counting of colony-forming units per urine millimeter inoculated in Petri plaque in CLED medium. The coincidence rate between both methods was 92,31, with sensitivity and specificity rates of 79,00% and 96,95% respectively. The level of sensitivity was affected by factors not directly dependent on the equipment. High values of specificity and of coincidence achieved by this equipment in relation to the reference method facilitates its use in urine culture, making possible to differentiate negative urine samples in 4 or 5 hours and to focus work and resources on positive samples.

  10. Sex and strain differences in the hepatocyte primary culture/DNA repair test

    SciTech Connect

    McQueen, C.A.; Way, B.M. )

    1991-01-01

    The hepatocyte primary culture (HPC)/DNA repair test was developed using hepatocytes isolated from male F-344 rats. A number of genetic polymorphisms have been shown to occur in inbred strains of rats, which may lead to variation in biotransformation of xenobiotics resulting in differences in susceptibility to genotoxins. The effect of the strain utilized as a source of hepatocytes was investigated with female Lewis, F-344, and DA rats. Variation was observed when hepatocytes from the three strains were exposed to aflatoxin B{sub 1} (AFB{sub 1}). No clearcut strain differences were seen when cells were exposed to diethylnitrosamine (DEN) or 2-acetylaminofluorene. These results demonstrate that both the strain and the sex of the animal used as a source of hepatocytes can affect the HPC/DNA repair test.

  11. Primary culture of intestinal epithelial cells as a potential model for Toxoplasma gondii enteric cycle studies.

    PubMed

    Moura, Marcos de Assis; Amendoeira, Maria Regina Reis; Barbosa, Helene Santos

    2009-09-01

    The primary culture of intestinal epithelial cells from domestic cats is an efficient cellular model to study the enteric cycle of Toxoplasma gondii in a definitive host. The parasite-host cell ratio can be pointed out as a decisive factor that determines the intracellular fate of bradyzoites forms. The development of the syncytial-like forms of T. gondii was observed using the 1:20 bradyzoite-host cell ratio, resulting in similar forms described in in vivo systems. This alternative study potentially opens up the field for investigation into the molecular aspects of this interaction. This can contribute to the development of new strategies for intervention of a main route by which toxoplasmosis spreads.

  12. Primary hepatocyte cultures as in vitro tools for toxicity testing: quo vadis?

    PubMed

    Vinken, Mathieu; Vanhaecke, Tamara; Rogiers, Vera

    2012-04-01

    Cultures of primary hepatocytes are versatile tools that can serve many in vitro toxicity testing purposes. However, they cope with dedifferentiation, a process that is already initiated during the hepatocyte isolation procedure and that is manifested as the progressive loss of functionality upon subsequent cultivation. A number of strategies to prevent dedifferentiation have been introduced over the last decades, all which aim at re-establishing the in vivo hepatocyte micro-environment in vitro, but that are of merely limited success. Recent mechanistic insight into the mechanisms that underlie hepatocyte dedifferentiation has opened new avenues for the development of novel approaches that target the actual causes of this deteriorative process and thus for the generation of a long-term hepatic in vitro tool. Such experimental system is urgently needed, especially in the light of the stringent European legislative modifications that are currently encountered by the pharmaceutical, chemical and, particularly, the cosmetic industry.

  13. Designing a primary science curriculum in a globalizing world: How do social constructivism and Vietnamese culture meet?

    NASA Astrophysics Data System (ADS)

    Hằng, Ngô Vũ Thu; Meijer, Marijn Roland; Bulte, Astrid M. W.; Pilot, Albert

    2016-03-01

    The implementation of social constructivist approaches to learning science in primary education in Vietnamese culture as an example of Confucian heritage culture remains challenging and problematic. This theoretical paper focuses on the initial phase of a design-based research approach; that is, the description of the design of a formal, written curriculum for primary science education in which features of social constructivist approaches to learning are synthesized with essential aspects of Vietnamese culture. The written design comprises learning aims, a framework that is the synthesis of learning functions, learning settings and educational expectations for learning phases, and exemplary curriculum units. Learning aims are formulated to comprehensively develop scientific knowledge, skills, and attitudes toward science for primary students. Derived from these learning aims, the designed framework consists of four learning phases respectively labeled as Engagement, Experience, Exchange, and Follow-up. The designed framework refers to knowledge of the "nature of science" education and characteristics of Vietnamese culture as an example of Confucian heritage culture. The curriculum design aims to serve as an educational product that addresses previously analyzed problems of primary science education in the Vietnamese culture in a globalizing world.

  14. MCP-1 expression by rat type II alveolar epithelial cells in primary culture.

    PubMed

    Paine, R; Rolfe, M W; Standiford, T J; Burdick, M D; Rollins, B J; Strieter, R M

    1993-05-15

    Recruitment and activation of mononuclear phagocytes are potentially critical regulatory events for control of pulmonary inflammation. Located at the boundary between the alveolar airspace and the interstitium, alveolar epithelial cells are ideally situated to regulate the recruitment and activation of mononuclear phagocytes through the production of cytokines in response to inflammatory stimulation from the alveolar space. To test this hypothesis, we investigated the production of monocyte chemotactic polypeptide-1 (MCP-1), a protein that is chemotactic for and that activates monocytes, by rat type II alveolar epithelial cells in primary culture. Immunocytochemical staining using anti-murine JE, an antibody recognizing rat MCP-1, demonstrated cell-associated MCP-1 Ag throughout the monolayer. The intensity of staining was increased in response to IL-1 beta. When type II epithelial cells formed a tight monolayer on a filter support, there was polar secretion of MCP-1 Ag into the apical compartment by both control and IL-1-stimulated cells as measured by specific MCP-1 ELISA. Northern blot analysis revealed that IL-1 and TNF-alpha stimulated MCP-1 mRNA expression in a dose-dependent manner, whereas dexamethasone blocked MCP-1 expression by cells stimulated with IL-1. In contrast to previous results using transformed epithelial cell lines, MCP-1 mRNA was induced in these primary cultures directly by stimulation with LPS. These data suggest that alveolar epithelial cells may have an important and previously unrecognized role in the initiation and maintenance of inflammatory processes in the lung by recruiting and activating circulating monocytes through the production of MCP-1.

  15. Excitatory amino acid-stimulated uptake of /sup 22/Na+ in primary astrocyte cultures

    SciTech Connect

    Kimelberg, H.K.; Pang, S.; Treble, D.H.

    1989-04-01

    In this study we have found that L-glutamic acid, as well as being taken up by a Na+-dependent mechanism, will stimulate the uptake of 22Na+ by primary astrocyte cultures from rat brain in the presence of ouabain. By simultaneously measuring the uptake of 22Na+ and L-3H-glutamate a stoichiometry of 2-3 Na+ per glutamate was measured, implying electrogenic uptake. Increasing the medium K+ concentration to depolarize the cells inhibited L-3H-glutamate uptake, while calculations of the energetics of the observed L-3H-glutamate accumulation also supported an electrogenic mechanism of at least 2 Na+:1 glutamate. In contrast, kinetic analysis of the Na+ dependence of L-3H-glutamate uptake indicated a stoichiometry of Na+ to glutamate of 1:1, but further analysis showed that the stoichiometry cannot be resolved by purely kinetic studies. Studies with glutamate analogs, however, showed that kainic acid was a very effective stimulant of 22Na+ uptake, but 3H-kainic acid showed no Na+ -dependent uptake. Furthermore, while L-3H-glutamate uptake was very sensitive to lowered temperatures, glutamate-stimulated 22Na+ uptake was relatively insensitive. These results indicate that glutamate-stimulated uptake of 22Na+ in primary astrocytes cultures cannot be explained solely by cotransport of Na+ with glutamate, and they suggest that direct kainic acid-type receptor induced stimulation of Na+ uptake also occurs. Since both receptor and uptake effects involve transport of Na+, accurate measurements of the Na+ :glutamate stoichiometry for uptake can only be done using completely specific inhibitors of these 2 systems.

  16. Primary neuron culture for nerve growth and axon guidance studies in zebrafish (Danio rerio).

    PubMed

    Chen, Zheyan; Lee, Han; Henle, Steven J; Cheever, Thomas R; Ekker, Stephen C; Henley, John R

    2013-01-01

    Zebrafish (Danio rerio) is a widely used model organism in genetics and developmental biology research. Genetic screens have proven useful for studying embryonic development of the nervous system in vivo, but in vitro studies utilizing zebrafish have been limited. Here, we introduce a robust zebrafish primary neuron culture system for functional nerve growth and guidance assays. Distinct classes of central nervous system neurons from the spinal cord, hindbrain, forebrain, and retina from wild type zebrafish, and fluorescent motor neurons from transgenic reporter zebrafish lines, were dissociated and plated onto various biological and synthetic substrates to optimize conditions for axon outgrowth. Time-lapse microscopy revealed dynamically moving growth cones at the tips of extending axons. The mean rate of axon extension in vitro was 21.4±1.2 µm hr(-1) s.e.m. for spinal cord neurons, which corresponds to the typical ∼0.5 mm day(-1) growth rate of nerves in vivo. Fluorescence labeling and confocal microscopy demonstrated that bundled microtubules project along axons to the growth cone central domain, with filamentous actin enriched in the growth cone peripheral domain. Importantly, the growth cone surface membrane expresses receptors for chemotropic factors, as detected by immunofluorescence microscopy. Live-cell functional assays of axon extension and directional guidance demonstrated mammalian brain-derived neurotrophic factor (BDNF)-dependent stimulation of outgrowth and growth cone chemoattraction, whereas mammalian myelin-associated glycoprotein inhibited outgrowth. High-resolution live-cell Ca(2+)-imaging revealed local elevation of cytoplasmic Ca(2+) concentration in the growth cone induced by BDNF application. Moreover, BDNF-induced axon outgrowth, but not basal outgrowth, was blocked by treatments to suppress cytoplasmic Ca(2+) signals. Thus, this primary neuron culture model system may be useful for studies of neuronal development, chemotropic axon

  17. Cellular metabolic rates in cultured primary dermal fibroblasts and myoblast cells from fast-growing and control Coturnix quail.

    PubMed

    Jimenez, Ana Gabriela; Cooper-Mullin, Clara; Anthony, Nicholas B; Williams, Joseph B

    2014-05-01

    Fibroblast cells have been extensively used in research, including in medicine, physiology, physiological-ecology, and conservation biology. However, whether the physiology of fibroblasts reflects the physiology of other cell types in the same animal is unknown. Dermal fibroblasts are responsible for generating connective tissue and involved in wound healing, but generally, this cell type is thought to be metabolically inactive until it is required at the site of tissue damage. Thus, one might question whether fibroblasts are a representative model system to portray the metabolic profile of the whole organism, as compared with cells isolated from other tissues, like muscle, brain or kidneys. To explore whether fibroblasts have the same metabolic profile as do myoblast cells, we cultured cells from day-old chicks of quail (Coturnix coturnix japonica) selected for fast-growth or normal growth (our control group). Our results suggest that isolated primary fibroblasts and myoblast cells had higher rates of glycolysis, oxygen consumption and more mitochondria in the fast-growing line than in the control line. Our findings lend support for the idea that fibroblasts are a representative cell system to characterize the whole organism metabolic signature at the cellular-level. These data are striking, however, because fibroblasts had higher rates of metabolism for every parameter measured than myoblast cells isolated from the same individuals.

  18. Forskolin delays the ethanol-induced desensitization of hypothalamic beta-endorphin neurons in primary cultures.

    PubMed

    Boyadjieva, N; Reddy, B V; Sarkar, D K

    1997-05-01

    Ethanol and its metabolite acetaldehyde have been shown to stimulate immunoreactive beta-endorphin (IR-beta-EP) secretion from hypothalamic neurons in primary cultures. Also, chronic ethanol and acetal-dehyde have been shown to cause the development of tolerance and desensitization of these neurons. In this study, we determined some of the cellular events leading to desensitization of the function of beta-endorphin (beta-EP) secretory neurons. The fetal hypothalamic cells were treated with various doses of ethanol (25 and 50 mM) or acetaldehyde (6.25, 12.5, and 25 mM) for 6 hr or treated with these drugs at 12 hr intervals for 72 hr. Determination of IR-beta-EP concentrations in the media revealed that ethanol increased IR-beta-EP secretion from these cultures for 12 hr, after this period, the cultured cells did not respond to ethanol. Acetaldehyde stimulated IR-beta-EP secretion from this culture for a period of 48 hr, but the IR-beta-EP secretory response to acetaldehyde reduced gradually with time during the first 48-hr period and reached the basal level at 72 hr. The desensitization of beta-EP neurons 12 hr after treatment with alcohol did not seem to be related to the loss of viable cells, because chronic ethanol exposures did not produce any effect on cell viability. However, reduced IR- beta-EP secretory response to acetaldehyde with time was associated with the time-dependent increase in cell death. Pretreatment of cultures with a cAMP analog, forskolin, increased the activity of functional beta-EP neurons and delayed the ethanol desensitization effects on these neurons. Pretreatment of forskolin did not delay the acetaldehyde desensitization of beta-EP neurons, but protected these cells from acetaldehyde toxicity. These results suggest that (i) chronic treatment with ethanol desensitizes beta-EP-secreting neurons due to reduced cellular functions and (ii) chronic acetaldehyde reduces beta-EP neurotransmission due to cell death. Furthermore, data suggest

  19. Differential Proteomic Analysis of Human Placenta-Derived Mesenchymal Stem Cells Cultured on Normal Tissue Culture Surface and Hyaluronan-Coated Surface

    PubMed Central

    Wong, Tzyy Yue; Chen, Ying-Hui; Liu, Szu-Heng; Solis, Mairim Alexandra; Yu, Chen-Hsiang; Chang, Chiung-Hsin; Huang, Lynn L. H.

    2016-01-01

    Our previous results showed that hyaluronan (HA) preserved human placenta-derived mesenchymal stem cells (PDMSC) in a slow cell cycling mode similar to quiescence, the pristine state of stem cells in vivo, and HA was found to prevent murine adipose-derived mesenchymal stem cells from senescence. Here, stable isotope labeling by amino acid in cell culture (SILAC) proteomic profiling was used to evaluate the effects of HA on aging phenomenon in stem cells, comparing (1) old and young passage PDMSC cultured on normal tissue culture surface (TCS); (2) old passage on HA-coated surface (CHA) compared to TCS; (3) old and young passage on CHA. The results indicated that senescence-associated protein transgelin (TAGLN) was upregulated in old TCS. Protein CYR61, reportedly senescence-related, was downregulated in old CHA compared to old TCS. The SIRT1-interacting Nicotinamide phosphoribosyltransferase (NAMPT) increased by 2.23-fold in old CHA compared to old TCS, and is 0.48-fold lower in old TCS compared to young TCS. Results also indicated that components of endoplasmic reticulum associated degradation (ERAD) pathway were upregulated in old CHA compared to old TCS cells, potentially for overcoming stress to maintain cell function and suppress senescence. Our data points to pathways that may be targeted by HA to maintain stem cells youth. PMID:27057169

  20. Melatonin Regulates Somatotrope and Lactotrope Function Through Common and Distinct Signaling Pathways in Cultured Primary Pituitary Cells From Female Primates

    PubMed Central

    Ibáñez-Costa, Alejandro; Córdoba-Chacón, José; Gahete, Manuel D.; Kineman, Rhonda D.; Castaño, Justo P.

    2015-01-01

    Melatonin (MT) is secreted by the pineal gland and exhibits a striking circadian rhythm in its release. Depending on the species studied, some pituitary hormones also display marked circadian/seasonal patterns and rhythms of secretion. However, the precise relationship between MT and pituitary function remains controversial, and studies focusing on the direct role of MT in normal pituitary cells are limited to nonprimate species. Here, adult normal primate (baboons) primary pituitary cell cultures were used to determine the direct impact of MT on the functioning of all pituitary cell types from the pars distalis. MT increased GH and prolactin (PRL) expression/release in a dose- and time-dependent fashion, a response that was blocked by somatostatin. However, MT did not significantly affect ACTH, FSH, LH, or TSH expression/release. MT did not alter GHRH- or ghrelin-induced GH and/or PRL secretions, suggesting that MT may activate similar signaling pathways as ghrelin/GHRH. The effects of MT on GH/PRL release, which are likely mediated through MT1 receptor, involve both common (adenylyl cyclase/protein kinase A/extracellular calcium-channels) and distinct (phospholipase C/intracellular calcium-channels) signaling pathways. Actions of MT on pituitary cells also included regulation of the expression of other key components for the control of somatotrope/lactotrope function (GHRH, ghrelin, and somatostatin receptors). These results show, for the first time in a primate model, that MT directly regulates somatotrope/lactotrope function, thereby lending support to the notion that the actions of MT on these cells might substantially contribute to the define daily patterns of GH and PRL observed in primates and perhaps in humans. PMID:25545385

  1. Sensitivity of primary fibroblasts in culture to atmospheric oxygen does not correlate with species lifespan

    PubMed Central

    Patrick, Alison; Seluanov, Michael; Hwang, Chaewon; Tam, Jonathan; Khan, Tanya; Morgenstern, Ari; Wiener, Lauren; Vazquez, Juan M.; Zafar, Hiba; Wen, Robert; Muratkalyeva, Malika; Doerig, Katherine; Zagorulya, Maria; Cole, Lauren; Catalano, Sophia; Lobo Ladd, Aliny AB; Coppi, A. Augusto; Coşkun, Yüksel; Tian, Xiao; Ablaeva, Julia; Nevo, Eviatar; Gladyshev, Vadim N.; Zhang, Zhengdong D.; Vijg, Jan; Seluanov, Andrei; Gorbunova, Vera

    2016-01-01

    Differences in the way human and mouse fibroblasts experience senescence in culture had long puzzled researchers. While senescence of human cells is mediated by telomere shortening, Parrinello et al. demonstrated that senescence of mouse cells is caused by extreme oxygen sensitivity. It was hypothesized that the striking difference in oxygen sensitivity between mouse and human cells explains their different rates of aging. To test if this hypothesis is broadly applicable, we cultured cells from 16 rodent species with diverse lifespans in 3% and 21% oxygen and compared their growth rates. Unexpectedly, fibroblasts derived from laboratory mouse strains were the only cells demonstrating extreme sensitivity to oxygen. Cells from hamster, muskrat, woodchuck, capybara, blind mole rat, paca, squirrel, beaver, naked mole rat and wild-caught mice were mildly sensitive to oxygen, while cells from rat, gerbil, deer mouse, chipmunk, guinea pig and chinchilla showed no difference in the growth rate between 3% and 21% oxygen. We conclude that, although the growth of primary fibroblasts is generally improved by maintaining cells in 3% oxygen, the extreme oxygen sensitivity is a peculiarity of laboratory mouse strains, possibly related to their very long telomeres, and fibroblast oxygen sensitivity does not directly correlate with species' lifespan. PMID:27163160

  2. Toxicity of organotin compounds in primary cultures of rat cortical astrocytes.

    PubMed

    Röhl, C; Gülden, M; Seibert, H

    2001-01-01

    The neurotoxic organotin compounds trimethyl (TMT) and triethyltin (TET) are known to induce astrogliosis in vivo, which is indicated by an increased synthesis of glial fibrillary acidic protein (GFAP) in astrocytes. In contrast, tributyltin (TBT) does not induce astrogliosis. The aim of this study was to investigate whether trialkyltin derivatives can induce an increased GFAP synthesis in astrocyte cultures in the absence of neurons and whether differences between the action of TMT, TET, and TBT can be detected. Primary cultures of rat cortical astrocytes from 2-day-old rats were grown in 96-well plates until confluency and then exposed to various concentrations of TMT, TET, and TBT for 40 h. Effects on basal cell functions were measured by colorimetric determination of cell protein contents and by assessment of viability by means of the MTT assay. An indirect sandwich ELISA for 96-well plates was used for quantitative measurements of the GFAP content of the cells. All three compounds induced a concentration-dependent cytotoxicity indicated by parallel decreases of protein contents and MTT reduction. Half-maximum cytotoxic concentrations were 3 micromol/L (TBT), 30 micromol/L (TET), and 800 micromol/L (TMT). Cellular GFAP contents were reduced in parallel to cytotoxic action but no increase in GFAP expression at subcytotoxic concentrations could be observed. Thus, the astrocytes were not able to respond to TMT or TET exposure by an increased synthesis of GFAP in the absence of neuronal signals.

  3. 3-bromopyruvate inhibits glycolysis, depletes cellular glutathione, and compromises the viability of cultured primary rat astrocytes.

    PubMed

    Ehrke, Eric; Arend, Christian; Dringen, Ralf

    2015-07-01

    The pyruvate analogue 3-bromopyruvate (3-BP) is an electrophilic alkylator that is considered a promising anticancer drug because it has been shown to kill cancer cells efficiently while having little toxic effect on nontumor cells. To test for potential adverse effects of 3-BP on brain cells, we exposed cultured primary rat astrocytes to 3-BP and investigated the effects of this compound on cell viability, glucose metabolism, and glutathione (GSH) content. The presence of 3-BP severely compromised cell viability and slowed cellular glucose consumption and lactate production in a time- and concentration-dependent manner, with half-maximal effects observed at about 100 µM 3-BP after 4 hr of incubation. The cellular hexokinase activity was not affected in 3-BP-treated astrocytes, whereas within 30 min after application of 3-BP the activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was inhibited, and cellular GSH content was depleted in a concentration-dependent manner, with half-maximal effects observed at about 30 µM 3-BP. The depletion of cellular GSH after exposure to 100 µM 3-BP was not prevented by the presence of 10 mM of the monocarboxylates lactate or pyruvate, suggesting that 3-BP is not taken up into astrocytes predominantly by monocarboxylate transporters. The data suggest that inhibition of glycolysis by inactivation of GAPDH and GSH depletion contributes to the toxicity that was observed for 3-BP-treated cultured astrocytes.

  4. [Primary culture and functional identification of distal pulmonary artery smooth muscle cells in mice].

    PubMed

    Li, M C; Chen, Y Q; Zhang, C T; Jiang, Q; Lu, W J; Wang, J

    2017-02-12

    Objective: To establish a method of isolation and primary culture of mice distal pulmonary artery smooth muscle cells (PASMCs) and identify the functional properties. Methods: PASMCs were harvested from the distal pulmonary artery (PA) tissue of mice by enzymatic digestion of collagenaseⅠand papain; and the growth characteristics were observed under inverted microscope and identified by Immunofluorescence technique. Effects on the intracellular calcium ion concentration of distal PASMCs were detected by Fura-2-AM fluorescent probe tracer under a fluorescence microscope in Krebs solution containing clopiazonic acid (CPA) and nifedipin (Nif). Results: PASMCs density reached approximately to 80% in a typical valley-peak-like shape after 6 days. Cell α-smooth muscle actin (α-SMA) immunofluorescence identified that 95% of the cultured cells were PASMCs. More than 95% PASMCs responded well to calcium-potassium Krebs solution (potassium ion concentration of 60 mmol/L) and showed a rapid increase in basal [Ca(2+) ](i) after 1 minute's perfusion (Δ[Ca(2+) ](i)>50), which demonstrated that the voltage-dependent calcium channels (VDCC) of distal PASMCs were in good function; after the perfusion of calcium Krebs, calcium-free/calcium-Krebs containing CPA and Nif, distal PASMCs showed two typical peaks, indicated the full function of store-operated calcium channel (SOCC) in distal PASMCs. Conclusion: This experiment successfully established a stable and reliable mice distal PASMCs model and the study of pulmonary vascular diseases could benefit from its higher purity and better functional condition.

  5. Antibody-Conjugated Single Quantum Dot Tracking of Membrane Neurotransmitter Transporters in Primary Neuronal Cultures.

    PubMed

    Bailey, Danielle M; Kovtun, Oleg; Rosenthal, Sandra J

    2017-01-01

    Single particle tracking (SPT) experiments have provided the scientific community with invaluable single-molecule information about the dynamic regulation of individual receptors, transporters, kinases, lipids, and molecular motors. SPT is an alternative to ensemble averaging approaches, where heterogeneous modes of motion might be lost. Quantum dots (QDs) are excellent probes for SPT experiments due to their photostability, high brightness, and size-dependent, narrow emission spectra. In a typical QD-based SPT experiment, QDs are bound to the target of interest and imaged for seconds to minutes via fluorescence video microscopy. Single QD spots in individual frames are then linked to form trajectories that are analyzed to determine their mean square displacement, diffusion coefficient, confinement index, and instantaneous velocity. This chapter describes a generalizable protocol for the single particle tracking of membrane neurotransmitter transporters on cell membranes with either unmodified extracellular antibody probes and secondary antibody-conjugated quantum dots or biotinylated extracellular antibody probes and streptavidin-conjugated quantum dots in primary neuronal cultures. The neuronal cell culture, the biotinylation protocol and the quantum dot labeling procedures, as well as basic data analysis are discussed.

  6. Effects of repetitive low-pressure explosive blast on primary neurons and mixed cultures.

    PubMed

    Zander, Nicole E; Piehler, Thuvan; Banton, Rohan; Benjamin, Richard

    2016-09-01

    Repetitive mild traumatic brain injury represents a considerable health concern, particularly for athletes and military personnel. For blast-induced brain injury, threshold shock-impulse levels required to induce such injuries and cumulative effects with single and/or multiple exposures are not well characterized. Currently, there is no established in vitro experimental model with blast pressure waves generated by live explosives. This study presents results of primary neurons and mixed cultures subjected to our unique in vitro indoor experimental platform that uses real military explosive charges to probe the effects of primary explosive blast at the cellular level. The effects of the blast on membrane permeability, generation of reactive oxygen species (ROS), uptake of sodium ions, intracellular calcium, and release of glutamate were probed 2 and 24 hr postblast. Significant changes in membrane permeability and sodium uptake among the sham, single-blast-injured, and triple-blast-injured samples were observed. A significant increase in ROS and glutamate release was observed for the triple-blast-injured samples compared with the sham. Changes in intracellular calcium were not significant. These results suggest that blast exposure disrupts the integrity of the plasma membrane, leading to the upset of ion homeostasis, formation of ROS, and glutamate release. Published 2016. †This article is a U.S. Government work and is in the public domain in the USA.

  7. Transcriptomic comparison of primary bovine horn core carcinoma culture and parental tissue at early stage

    PubMed Central

    Shil, Sharadindu; Joshi, R. S.; Joshi, C. G.; Patel, A. K.; Shah, Ravi K.; Patel, Namrata; Jakhesara, Subhash J.; Kundu, Sumana; Reddy, Bhaskar; Koringa, P. G.; Rank, D. N.

    2017-01-01

    Aim: Squamous cell carcinoma or SCC of horn in bovines (bovine horn core carcinoma) frequently observed in Bos indicus affecting almost 1% of cattle population. Freshly isolated primary epithelial cells may be closely related to the malignant epithelial cells of the tumor. Comparison of gene expression in between horn’s SCC tissue and its early passage primary culture using next generation sequencing was the aim of this study. Materials and Methods: Whole transcriptome sequencing of horn’s SCC tissue and its early passage cells using Ion Torrent PGM were done. Comparative expression and analysis of different genes and pathways related to cancer and biological processes associated with malignancy, proliferating capacity, differentiation, apoptosis, senescence, adhesion, cohesion, migration, invasion, angiogenesis, and metabolic pathways were identified. Results: Up-regulated genes in SCC of horn’s early passage cells were involved in transporter activity, catalytic activity, nucleic acid binding transcription factor activity, biogenesis, cellular processes, biological regulation and localization and the down-regulated genes mainly were involved in focal adhesion, extracellular matrix receptor interaction and spliceosome activity. Conclusion: The experiment revealed similar transcriptomic nature of horn’s SCC tissue and its early passage cells. PMID:28246447

  8. Axenic in vitro cultivation of Echinococcus multilocularis metacestode vesicles and the generation of primary cell cultures.

    PubMed

    Spiliotis, Markus; Brehm, Klaus

    2009-01-01

    Parasitic helminths are a major cause of disease worldwide, yet the molecular mechanisms of host-helminth interaction and parasite development are only rudimentarily studied. A main reasons for this lack of knowledge are the tremendous experimental difficulties in cultivating parasitic helminths under defined laboratory conditions and obtaining sufficient amounts of parasite material for molecular analyses. For one member of this neglected group of pathogens, the fox-tapeworm Echinococcus multilocularis, we have established and optimized in vitro cultivation systems by which the major part of the parasite's life cycle, leading from early metacestode vesicles to the production of protoscoleces, can be mimicked under laboratory conditions. The methodology comprises co-cultivation systems for host cells and parasite larvae by which large amounts of parasite vesicles can be generated. Furthermore, we have established an axenic (host cell-free) cultivation system that allows studies on the influence of defined host factors on parasite growth and development. On the basis of this system, the isolation and maintenance of primary Echinococcus cells that are devoid of overgrowing host cells is now possible. The availability of the primary cell culture system constitutes a first step toward the establishment of genetic manipulation methods for the parasite that will be of great interest for further research on infection strategies and development of Echinococcus and other cestodes.

  9. Sulfuretin promotes osteoblastic differentiation in primary cultured osteoblasts and in vivo bone healing

    PubMed Central

    Yun, Hyung-Mun; Lim, Hyun-Chang; Kim, Ga-Hyun; Lee, Dong-Sung; Kim, Youn-Chul; Oh, Hyuncheol; Kim, Eun-Cheol

    2016-01-01

    Although sulfuretin, the major flavonoid of Rhus verniciflua Stokes, has a variety of biological actions, its in vitro and in vivo effects on osteogenic potential remain poorly understood. The objective of the present study was to investigate the effects of sulfuretin on in vitro osteoblastic differentiation and the underlying signal pathway mechanisms in primary cultured osteoblasts and on in vivo bone formation using critical-sized calvarial defects in mice. Sulfuretin promoted osteogenic differentiation of primary osteoblasts, with increased ALP activity and mineralization, and upregulated differentiation markers, including ALP, osteocalcin, and osteopontin, in a concentration-dependent manner. The expression levels of Runx2, BMP-2, and phospho-Smad1/5/8 were upregulated by sulfuretin. Moreover, sulfuretin increased phosphorylation of Akt, mTOR, ERK, and JNK. Furthermore, sulfuretin treatment increased mRNA expression of Wnt ligands, phosphorylation of GSK3, and nuclear β-catenin protein expression. In vivo studies with calvarial bone defects revealed that sulfuretin significantly enhanced new bone formation by micro-computed tomography and histologic analysis. Collectively, these data suggest that sulfuretin acts through the activation of BMP, mTOR, Wnt/β-catenin, and Runx2 signaling to promote in vitro osteoblast differentiation and facilitate in vivo bone regeneration, and might be have therapeutic benefits in bone disease and regeneration. PMID:27713171

  10. Preparation of Primary Myogenic Precursor Cell/Myoblast Cultures from Basal Vertebrate Lineages

    PubMed Central

    Froehlich, Jacob Michael; Seiliez, Iban; Gabillard, Jean-Charles; Biga, Peggy R.

    2014-01-01

    Due to the inherent difficulty and time involved with studying the myogenic program in vivo, primary culture systems derived from the resident adult stem cells of skeletal muscle, the myogenic precursor cells (MPCs), have proven indispensible to our understanding of mammalian skeletal muscle development and growth. Particularly among the basal taxa of Vertebrata, however, data are limited describing the molecular mechanisms controlling the self-renewal, proliferation, and differentiation of MPCs. Of particular interest are potential mechanisms that underlie the ability of basal vertebrates to undergo considerable postlarval skeletal myofiber hyperplasia (i.e. teleost fish) and full regeneration following appendage loss (i.e. urodele amphibians). Additionally, the use of cultured myoblasts could aid in the understanding of regeneration and the recapitulation of the myogenic program and the differences between them. To this end, we describe in detail a robust and efficient protocol (and variations therein) for isolating and maintaining MPCs and their progeny, myoblasts and immature myotubes, in cell culture as a platform for understanding the evolution of the myogenic program, beginning with the more basal vertebrates. Capitalizing on the model organism status of the zebrafish (Danio rerio), we report on the application of this protocol to small fishes of the cyprinid clade Danioninae. In tandem, this protocol can be utilized to realize a broader comparative approach by isolating MPCs from the Mexican axolotl (Ambystomamexicanum) and even laboratory rodents. This protocol is now widely used in studying myogenesis in several fish species, including rainbow trout, salmon, and sea bream1-4. PMID:24835774

  11. Cytogenetic aberrations in primary cell cultures of the ovarian surface epithelium.

    PubMed

    Chuaire-Noack, Lilian; Rondón-Lagos, Sandra; Ramírez-Corredor, Amparo; Ibáñez-Pinilla, Milcíades; Ramírez-Clavijo, Sandra

    2010-12-01

    Our objective was to determine the presence of chromosomal abnormalities in primary cultures of ovarian surface epithelial cells in women of different ages with no history of cancer. Throughout conventional cytogenetic techniques, we analyzed chromosome spreads of cultured ovarian epithelial cells from 10 donors who were 50 or more years old (B) and 16 controls between 20 and 49 years old (A), belonging to the mestizo population in Bogota DC, Colombia. Of the 26 cultures that were analyzed in passage 1, 61.5% had an abnormal chromosome complement (62.5% in A, and 60% in B). Abnormalities included polyploidies, endoduplications and monosomies. Deletions in chromosomes 3 and 11 were found in just one metaphase. None of the samples showed weaknesses or breakpoints. After transforming and applying the exact student's t-test for variance heterogeneity, we found significant differences in the frequency of metaphases, that were higher in A than in B (p=0.05), and in the frequency of polyploidies, which were higher in B than in A (p=0.044). Through the application of the Mann-Whitney test, we determined that the frequency of endoduplications was higher in A than in B (p=0.126), without reaching significant differences. There were no significant differences in the frequency of monosomies. The level of significance was set at p < or = 0.05. Taking into account that polyploidization is a marker of chromosomal instability and that the risk of cancer arising from the ovarian surface epithelium augments substantially after menopause, the increase in the frequency of age-associated polyploidies could be used as a predictor of ovarian cancer in women from an ethnically homogeneous population as the mestizo one in Bogota DC.

  12. Prognostic and biologic significance of DNMT3B expression in older patients with cytogenetically normal primary acute myeloid leukemia.

    PubMed

    Niederwieser, C; Kohlschmidt, J; Volinia, S; Whitman, S P; Metzeler, K H; Eisfeld, A-K; Maharry, K; Yan, P; Frankhouser, D; Becker, H; Schwind, S; Carroll, A J; Nicolet, D; Mendler, J H; Curfman, J P; Wu, Y-Z; Baer, M R; Powell, B L; Kolitz, J E; Moore, J O; Carter, T H; Bundschuh, R; Larson, R A; Stone, R M; Mrózek, K; Marcucci, G; Bloomfield, C D

    2015-03-01

    DNMT3B encodes a DNA methyltransferase implicated in aberrant epigenetic changes contributing to leukemogenesis. We tested whether DNMT3B expression, measured by NanoString nCounter assay, associates with outcome, gene and microRNA expression and DNA methylation profiles in 210 older (⩾60 years) adults with primary, cytogenetically normal acute myeloid leukemia (CN-AML). Patients were dichotomized into high versus low expressers using median cut. Outcomes were assessed in the context of known CN-AML prognosticators. Gene and microRNA expression, and DNA methylation profiles were analyzed using microarrays and MethylCap-sequencing, respectively. High DNMT3B expressers had fewer complete remissions (CR; P=0.002) and shorter disease-free (DFS; P=0.02) and overall (OS; P<0.001) survival. In multivariable analyses, high DNMT3B expression remained an independent predictor of lower CR rates (P=0.04) and shorter DFS (P=0.04) and OS (P=0.001). High DNMT3B expression associated with a gene expression profile comprising 363 genes involved in differentiation, proliferation and survival pathways, but with only four differentially expressed microRNAs (miR-133b, miR-148a, miR-122, miR-409-3p) and no differential DNA methylation regions. We conclude that high DNMT3B expression independently associates with adverse outcome in older CN-AML patients. Gene expression analyses suggest that DNMT3B is involved in the modulation of several genes, although the regulatory mechanisms remain to be investigated to devise therapeutic approaches specific for these patients.

  13. Propagation of normal human epithelial cell populations using an in vivo culture system. Description and applications.

    PubMed Central

    Klein-Szanto, A. J.; Terzaghi, M.; Mirkin, L. D.; Martin, D.; Shiba, M.

    1982-01-01

    A new model using xenotransplanted human epithelia was developed for the study of toxic and carcinogenic effects of chemicals. Epithelial cells from the respiratory tract of 4 male and 3 female premature and fullterm fetuses were enzymatically removed and inoculated into deepithelialized rat tracheas. These were sealed at both ends and transplanted subcutaneously into nude mice. After 3-4 weeks, a normal mucociliary epithelium covered the tracheal lumen. At this stage the epithelial cells could be isolated again and transplanted into new denuded rat tracheas. This passaging could be repeated up to six times, each permitting an amplification factor of approximately 3. Tracheal transplants containing cells of human origin (in vivo Passages 2-4) were treated with 7,12-dimethylbenz(a)anthracene. Hyperplasias, squamous metaplasias, and dysplasias were seen 1-8 weeks after initiation of treatment, indicating that the responses of human and rodent epithelial cells to polycyclic aromatic hydrocarbons are similar. Initial experiments with skin and esophageal epithelia suggest that other covering epithelia could also be used in this fashion for evaluation of toxicants and carcinogens that are likely to come into contact with these tissues. Images Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 PMID:6821529

  14. Innovative approaches to establish and characterize primary cultures: an ex vivo 3D system and the zebrafish model

    PubMed Central

    Liverani, Chiara; La Manna, Federico; Groenewoud, Arwin; Mercatali, Laura; Van Der Pluijm, Gabri; Pieri, Federica; Cavaliere, Davide; De Vita, Alessandro; Spadazzi, Chiara; Miserocchi, Giacomo; Bongiovanni, Alberto; Recine, Federica; Riva, Nada; Amadori, Dino; Tasciotti, Ennio; Snaar-Jagalska, Ewa

    2017-01-01

    ABSTRACT Patient-derived specimens are an invaluable resource to investigate tumor biology. However, in vivo studies on primary cultures are often limited by the small amount of material available, while conventional in vitro systems might alter the features and behavior that characterize cancer cells. We present our data obtained on primary dedifferentiated liposarcoma cells cultured in a 3D scaffold-based system and injected into a zebrafish model. Primary cells were characterized in vitro for their morphological features, sensitivity to drugs and biomarker expression, and in vivo for their engraftment and invasiveness abilities. The 3D culture showed a higher enrichment in cancer cells than the standard monolayer culture and a better preservation of liposarcoma-associated markers. We also successfully grafted primary cells into zebrafish, showing their local migratory and invasive abilities. Our work provides proof of concept of the ability of 3D cultures to maintain the original phenotype of ex vivo cells, and highlights the potential of the zebrafish model to provide a versatile in vivo system for studies with limited biological material. Such models could be used in translational research studies for biomolecular analyses, drug screenings and tumor aggressiveness assays. PMID:27895047

  15. Feasibility of Primary Tumor Culture Models and Preclinical Prediction Assays for Head and Neck Cancer: A Narrative Review

    PubMed Central

    Dohmen, Amy J. C.; Swartz, Justin E.; Van Den Brekel, Michiel W. M.; Willems, Stefan M.; Spijker, René; Neefjes, Jacques; Zuur, Charlotte L.

    2015-01-01

    Primary human tumor culture models allow for individualized drug sensitivity testing and are therefore a promising technique to achieve personalized treatment for cancer patients. This would especially be of interest for patients with advanced stage head and neck cancer. They are extensively treated with surgery, usually in combination with high-dose cisplatin chemoradiation. However, adding cisplatin to radiotherapy is associated with an increase in severe acute toxicity, while conferring only a minor overall survival benefit. Hence, there is a strong need for a preclinical model to identify patients that will respond to the intended treatment regimen and to test novel drugs. One of such models is the technique of culturing primary human tumor tissue. This review discusses the feasibility and success rate of existing primary head and neck tumor culturing techniques and their corresponding chemo- and radiosensitivity assays. A comprehensive literature search was performed and success factors for culturing in vitro are debated, together with the actual value of these models as preclinical prediction assay for individual patients. With this review, we aim to fill a gap in the understanding of primary culture models from head and neck tumors, with potential importance for other tumor types as well. PMID:26343729

  16. Generation of viable embryos and embryonic stem cell-like cells from cultured primary follicles in mice.

    PubMed

    Choi, Jun Hee; Kim, Gil Ah; Park, Jong Heum; Song, Gwon Hwa; Park, Jun Won; Kim, Dae Yong; Lim, Jeong Mook

    2011-10-01

    Primary follicles retrieved from B6CBAF1 prepubertal mice were cultured in a stepwise manner in an alpha-minimum essential medium-based medium to generate viable embryos and embryonic stem cell (ESC)-like cells. A significant increase in follicle growth and oocyte maturation accompanied by increased secretion of 17beta-estradiol and progesterone was achieved by exposing primary follicles to 100 or 200 mIU of follicle-stimulating hormone (FSH) during culture. More oocytes developed into blastocysts following in vitro fertilization (IVF) or parthenogenetic activation after culture with 200 mIU of FSH during the entire culture period than with 100 mIU. Eleven ESC-like cell lines, consisting of four heterozygotic and seven homozygotic phenotypes, were established from 25 trials of primary follicle culture combined with IVF or parthenogenetic activation. In conclusion, primary follicles can potentially yield developmentally competent oocytes, which produce viable embryos and ESC-like cell lines following in vitro manipulation. We suggest a method to utilize immature follicles, which are most abundant in ovaries, to improve reproductive efficiency and for use in regenerative medicine.

  17. Integrated economic and experimental framework for screening of primary recovery technologies for high cell density CHO cultures

    PubMed Central

    Popova, Daria; Stonier, Adam; Pain, David; Titchener‐Hooker, Nigel J.

    2016-01-01

    Abstract Increases in mammalian cell culture titres and densities have placed significant demands on primary recovery operation performance. This article presents a methodology which aims to screen rapidly and evaluate primary recovery technologies for their scope for technically feasible and cost‐effective operation in the context of high cell density mammalian cell cultures. It was applied to assess the performance of current (centrifugation and depth filtration options) and alternative (tangential flow filtration (TFF)) primary recovery strategies. Cell culture test materials (CCTM) were generated to simulate the most demanding cell culture conditions selected as a screening challenge for the technologies. The performance of these technology options was assessed using lab scale and ultra scale‐down (USD) mimics requiring 25–110mL volumes for centrifugation and depth filtration and TFF screening experiments respectively. A centrifugation and depth filtration combination as well as both of the alternative technologies met the performance selection criteria. A detailed process economics evaluation was carried out at three scales of manufacturing (2,000L, 10,000L, 20,000L), where alternative primary recovery options were shown to potentially provide a more cost‐effective primary recovery process in the future. This assessment process and the study results can aid technology selection to identify the most effective option for a specific scenario. PMID:27067803

  18. Hyaline cartilage tissue is formed through the co-culture of passaged human chondrocytes and primary bovine chondrocytes.

    PubMed

    Taylor, Drew W; Ahmed, Nazish; Hayes, Anthony J; Ferguson, Peter; Gross, Allan E; Caterson, Bruce; Kandel, Rita A

    2012-08-01

    To circumvent the problem of a sufficient number of cells for cartilage engineering, the authors previously developed a two-stage culture system to redifferentiate monolayer culture-expanded dedifferentiated human articular chondrocytes by co-culture with primary bovine chondrocytes (bP0). The aim of this study was to analyze the composition of the cartilage tissue formed in stage 1 and compare it with bP0 grown alone to determine the optimal length of the co-culture stage of the system. Biochemical data show that extracellular matrix accumulation was evident after 2 weeks of co-culture, which was 1 week behind the bP0 control culture. By 3 to 4 weeks, the amounts of accumulated proteoglycans and collagens were comparable. Expression of chondrogenic genes, Sox 9, aggrecan, and collagen type II, was also at similar levels by week 3 of culture. Immunohistochemical staining of both co-culture and control tissues showed accumulation of type II collagen, aggrecan, biglycan, decorin, and chondroitin sulfate in appropriate zonal distributions. These data indicate that co-cultured cells form cartilaginous tissue that starts to resemble that formed by bP0 after 3 weeks, suggesting that the optimal time to terminate the co-culture stage, isolate the now redifferentiated cells, and start stage 2 is just after 3 weeks.

  19. Effect of culture conditions on microRNA expression in primary adult control and COPD lung fibroblasts in vitro.

    PubMed

    Ikari, Jun; Smith, Lynette M; Nelson, Amy J; Iwasawa, Shunichiro; Gunji, Yoko; Farid, Maha; Wang, Xingqi; Basma, Hesham; Feghali-Bostwick, Carol; Liu, Xiangde; DeMeo, Dawn L; Rennard, Stephen I

    2015-04-01

    In vitro cell cultures, including lung fibroblasts, have been used to identify microRNAs (miRNAs) associated with chronic obstructive pulmonary disease (COPD) pathogenesis. However, culture conditions may affect miRNA expression. We examined whether miRNA expression in primary adult lung fibroblasts varies with cell density or passage in vitro and whether culture conditions confound the identification of altered miRNA expression in COPD lung fibroblasts. Primary adult control and COPD lung fibroblasts were cultured until passage 3 or 8, after which cells were further cultured for 3 or 7 d (low vs. high density). Then, cells at low density were cultured with serum-free media, and those at high density were cultured with serum-free media in the absence or presence of interleukin-1β (IL-1β) and tumor necrosis factor alpha (TNF-α) for 24 h. RNA was extracted to perform miRNA microarray from which 1.25-fold differential expression and 10% false discovery rate were applied to identify "invariant" and "variant" miRNA for the various culture conditions. Of the 2226 miRNAs evaluated, 39.0% for cell density, 40.7% for cell passage, and 29.4% for both conditions were identified as "invariant" miRNAs. Furthermore, 38.1% of the evaluated miRNAs were "invariant" for cell passage with IL-1β and TNF-α. Differentially expressed miRNAs between control and COPD lung fibroblasts were identified with and without IL-1β and TNF-α, and of these, 32 out of the 34 top-ranked miRNAs exceeded the differences due to culture conditions. Thus, culture conditions may affect miRNA expression of adult human lung fibroblasts. Nevertheless, in vitro cultures can be used to assess differential miRNA expression in COPD lung fibroblasts.

  20. Primary Human Uterine Leiomyoma Cell Culture Quality Control: Some Properties of Myometrial Cells Cultured under Serum Deprivation Conditions in the Presence of Ovarian Steroids

    PubMed Central

    Sumikawa, Joana Tomomi; Batista, Fabrício Pereira; Paredes-Gamero, Edgar J.; Girão, Manoel J. B. C.; Oliva, Maria Luiza V.

    2016-01-01

    Cell culture is considered the standard media used in research to emulate the in vivo cell environment. Crucial in vivo experiments cannot be conducted in humans and depend on in vitro methodologies such as cell culture systems. However, some procedures involving the quality control of cells in culture have been gradually neglected by failing to acknowledge that primary cells and cell lines change over time in culture. Thus, we report methods based on our experience for monitoring primary cell culture of human myometrial cells derived from uterine leiomyoma. We standardized the best procedure of tissue dissociation required for the study of multiple genetic marker systems that include species-specific antigens, expression of myofibroblast or myoblast markers, growth curve, serum deprivation, starvation by cell cycle synchronization, culture on collagen coated plates, and 17 β-estradiol (E2) and progesterone (P4) effects. The results showed that primary myometrial cells from patients with uterine leiomyoma displayed myoblast phenotypes before and after in vitro cultivation, and leiomyoma cells differentiated into mature myocyte cells under the appropriate differentiation-inducing conditions (serum deprivation). These cells grew well on collagen coated plates and responded to E2 and P4, which may drive myometrial and leiomyoma cells to proliferate and adhere into a focal adhesion complex involvement in a paracrine manner. The establishment of these techniques as routine procedures will improve the understanding of the myometrial physiology and pathogenesis of myometrium-derived diseases such as leiomyoma. Mimicking the in vivo environment of fibrotic conditions can prevent false results and enhance results that are based on cell culture integrity. PMID:27391384

  1. Temperature rise under normal and caries-affected primary tooth dentin disks during polymerization of adhesives and resin-containing dental materials.

    PubMed

    Tosun, Gul; Usumez, Aslihan; Yondem, Isa; Sener, Yagmur

    2008-05-01

    The purpose of this study was to compare the temperature rise under normal and caries-affected primary tooth dentin during photopolymerization of two adhesives and resin-containing restorative materials. Caries-affected and normal dentin disks were prepared from extracted primary molars with only mesial or distal approximal caries (4 mm in diameter, 1 mm in height). Temperature rise during photopolymerization of adhesive materials was measured with a J-type thermocouple wire that was connected to a data logger. Data were analyzed with two-way ANOVA and independent samples t-test. Temperature rise under caries-affected primary tooth dentin disks was higher than that of normal primary tooth dentin disks during polymerization of both adhesive systems and resin-containing dental materials (p < 0.05). It was found that adhesive systems induced a higher temperature rise during polymerization as compared to the resin-containing restorative materials (p < 0.05). In particular, temperature rise during polymerization of adhesive materials exceeded 5.5 degrees C under caries-affected primary tooth dentin.

  2. Intracellular degradation of chemically functionalized carbon nanotubes using a long-term primary microglial culture model

    NASA Astrophysics Data System (ADS)

    Bussy, Cyrill; Hadad, Caroline; Prato, Maurizio; Bianco, Alberto; Kostarelos, Kostas

    2015-12-01

    Chemically functionalized carbon nanotubes (f-CNTs) have been used in proof-of-concept studies to alleviate debilitating neurological conditions. Previous in vivo observations in brain tissue have suggested that microglia - acting as resident macrophages of the brain - play a critical role in the internalization of f-CNTs and their partial in situ biodegradation following a stereotactic administration in the cortex. At the same time, several reports have indicated that immune cells such as neutrophils, eosinophils and even macrophages could participate in the processing of carbon nanomaterials via oxidation processes leading to degradation, with surface properties acting as modulators of CNT biodegradability. In this study we questioned whether degradability of f-CNTs within microglia could be modulated depending on the type of surface functionalization used. We investigated the kinetics of degradation of multi-walled carbon nanotubes (MWNTs) functionalized via different chemical strategies that were internalized within isolated primary microglia over three months. A cellular model of rat primary microglia that can be maintained in cell culture for a long period of time was first developed. The Raman structural signature of the internalized f-CNTs was then studied directly in cells over a period of up to three months, following a single exposure to a non-cytotoxic concentration of three different f-CNTs (carboxylated, aminated and both carboxylated and aminated). Structural modifications suggesting partial but continuous degradation were observed for all nanotubes irrespective of their surface functionalization. Carboxylation was shown to promote more pronounced structural changes inside microglia over the first two weeks of the study.Chemically functionalized carbon nanotubes (f-CNTs) have been used in proof-of-concept studies to alleviate debilitating neurological conditions. Previous in vivo observations in brain tissue have suggested that microglia - acting as

  3. Intracellular cholesterol-binding proteins enhance HDL-mediated cholesterol uptake in cultured primary mouse hepatocytes

    PubMed Central

    Storey, Stephen M.; McIntosh, Avery L.; Huang, Huan; Landrock, Kerstin K.; Martin, Gregory G.; Landrock, Danilo; Payne, H. Ross; Atshaves, Barbara P.; Kier, Ann B.

    2012-01-01

    A major gap in our knowledge of rapid hepatic HDL cholesterol clearance is the role of key intracellular factors that influence this process. Although the reverse cholesterol transport pathway targets HDL to the liver for net elimination of free cholesterol from the body, molecular details governing cholesterol uptake into hepatocytes are not completely understood. Therefore, the effects of sterol carrier protein (SCP)-2 and liver fatty acid-binding protein (L-FABP), high-affinity cholesterol-binding proteins present in hepatocyte cytosol, on HDL-mediated free cholesterol uptake were examined using gene-targeted mouse models, cultured primary hepatocytes, and 22-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-amino]-23,24-bisnor-5-cholen-3β-ol (NBD-cholesterol). While SCP-2 overexpression enhanced NBD-cholesterol uptake, counterintuitively, SCP-2/SCP-x gene ablation also 1) enhanced the rapid molecular phase of free sterol uptake detectable in <1 min and initial rate and maximal uptake of HDL free cholesterol and 2) differentially enhanced free cholesterol uptake mediated by the HDL3, rather than the HDL2, subfraction. The increased HDL free cholesterol uptake was not due to increased expression or distribution of the HDL receptor [scavenger receptor B1 (SRB1)], proteins regulating SRB1 [postsynaptic density protein (PSD-95)/Drosophila disk large tumor suppressor (dlg)/tight junction protein (ZO1) and 17-kDa membrane-associated protein], or other intracellular cholesterol trafficking proteins (steroidogenic acute response protein D, Niemann Pick C, and oxysterol-binding protein-related proteins). However, expression of L-FABP, the single most prevalent hepatic cytosolic protein that binds cholesterol, was upregulated twofold in SCP-2/SCP-x null hepatocytes. Double-immunogold electron microscopy detected L-FABP sufficiently close to SRB1 for direct interaction, similar to SCP-2. These data suggest a role for L-FABP in HDL cholesterol uptake, a finding confirmed with SCP-2

  4. Intracellular cholesterol-binding proteins enhance HDL-mediated cholesterol uptake in cultured primary mouse hepatocytes.

    PubMed

    Storey, Stephen M; McIntosh, Avery L; Huang, Huan; Landrock, Kerstin K; Martin, Gregory G; Landrock, Danilo; Payne, H Ross; Atshaves, Barbara P; Kier, Ann B; Schroeder, Friedhelm

    2012-04-15

    A major gap in our knowledge of rapid hepatic HDL cholesterol clearance is the role of key intracellular factors that influence this process. Although the reverse cholesterol transport pathway targets HDL to the liver for net elimination of free cholesterol from the body, molecular details governing cholesterol uptake into hepatocytes are not completely understood. Therefore, the effects of sterol carrier protein (SCP)-2 and liver fatty acid-binding protein (L-FABP), high-affinity cholesterol-binding proteins present in hepatocyte cytosol, on HDL-mediated free cholesterol uptake were examined using gene-targeted mouse models, cultured primary hepatocytes, and 22-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-amino]-23,24-bisnor-5-cholen-3β-ol (NBD-cholesterol). While SCP-2 overexpression enhanced NBD-cholesterol uptake, counterintuitively, SCP-2/SCP-x gene ablation also 1) enhanced the rapid molecular phase of free sterol uptake detectable in <1 min and initial rate and maximal uptake of HDL free cholesterol and 2) differentially enhanced free cholesterol uptake mediated by the HDL3, rather than the HDL2, subfraction. The increased HDL free cholesterol uptake was not due to increased expression or distribution of the HDL receptor [scavenger receptor B1 (SRB1)], proteins regulating SRB1 [postsynaptic density protein (PSD-95)/Drosophila disk large tumor suppressor (dlg)/tight junction protein (ZO1) and 17-kDa membrane-associated protein], or other intracellular cholesterol trafficking proteins (steroidogenic acute response protein D, Niemann Pick C, and oxysterol-binding protein-related proteins). However, expression of L-FABP, the single most prevalent hepatic cytosolic protein that binds cholesterol, was upregulated twofold in SCP-2/SCP-x null hepatocytes. Double-immunogold electron microscopy detected L-FABP sufficiently close to SRB1 for direct interaction, similar to SCP-2. These data suggest a role for L-FABP in HDL cholesterol uptake, a finding confirmed with SCP-2

  5. The neurotoxicity of hallucinogenic amphetamines in primary cultures of hippocampal neurons.

    PubMed

    Capela, João Paulo; da Costa Araújo, Silvana; Costa, Vera Marisa; Ruscher, Karsten; Fernandes, Eduarda; Bastos, Maria de Lourdes; Dirnagl, Ulrich; Meisel, Andreas; Carvalho, Félix

    2013-01-01

    3,4-Methylenedioxymethamphetamine (MDMA or "Ecstasy") and 2,5-dimethoxy-4-iodoamphetamine hydrochloride (DOI) are hallucinogenic amphetamines with addictive properties. The hippocampus is involved in learning and memory and seems particularly vulnerable to amphetamine's neurotoxicity. We evaluated the neurotoxicity of DOI and MDMA in primary neuronal cultures of hippocampus obtained from Wistar rat embryos (E-17 to E-19). Mature neurons after 10 days in culture were exposed for 24 or 48 h either to MDMA (100-800 μM) or DOI (10-100 μM). Both the lactate dehydrogenase (LDH) release and the tetrazolium-based (MTT) assays revealed a concentration- and time-dependent neuronal death and mitochondrial dysfunction after exposure to both drugs. Both drugs promoted a significant increase in caspase-8 and caspase-3 activities. At concentrations that produced similar levels of neuronal death, DOI promoted a higher increase in the activity of both caspases than MDMA. In the mitochondrial fraction of neurons exposed 24h to DOI or MDMA, we found a significant increase in the 67 kDa band of apoptosis inducing factor (AIF) by Western blot. Moreover, 24h exposure to DOI promoted an increase in cytochrome c in the cytoplasmatic fraction of neurons. Pre-treatment with an antibody raised against the 5-HT(2A)-receptor (an irreversible antagonist) greatly attenuated neuronal death promoted by 48 h exposure to DOI or MDMA. In conclusion, hallucinogenic amphetamines promoted programmed neuronal death involving both the mitochondria machinery and the extrinsic cell death key regulators. Death was dependent, at least in part, on the stimulation of the 5-HT(2A)-receptors.

  6. Development of a selective culture medium for primary isolation of the main Brucella species.

    PubMed

    De Miguel, M J; Marín, C M; Muñoz, P M; Dieste, L; Grilló, M J; Blasco, J M

    2011-04-01

    Bacteriological diagnosis of brucellosis is performed by culturing animal samples directly on both Farrell medium (FM) and modified Thayer-Martin medium (mTM). However, despite inhibiting most contaminating microorganisms, FM also inhibits the growth of Brucella ovis and some B. melitensis and B. abortus strains. In contrast, mTM is adequate for growth of all Brucella species but only partially inhibitory for contaminants. Moreover, the performance of both culture media for isolating B. suis has never been established properly. We first determined the performance of both media for B. suis isolation, proving that FM significantly inhibits B. suis growth. We also determined the susceptibility of B. suis to the antibiotics contained in both selective media, proving that nalidixic acid and bacitracin are highly inhibitory, thus explaining the reduced performance of FM for B. suis isolation. Based on these results, a new selective medium (CITA) containing vancomycin, colistin, nystatin, nitrofurantoin, and amphotericin B was tested for isolation of the main Brucella species, including B. suis. CITA's performance was evaluated using reference contaminant strains but also field samples taken from brucella-infected animals or animals suspected of infection. CITA inhibited most contaminant microorganisms but allowed the growth of all Brucella species, to levels similar to those for both the control medium without antibiotics and mTM. Moreover, CITA medium was more sensitive than both mTM and FM for isolating all Brucella species from field samples. Altogether, these results demonstrate the adequate performance of CITA medium for the primary isolation of the main Brucella species, including B. suis.

  7. Copper Nanoparticles and Copper Sulphate Induced Cytotoxicity in Hepatocyte Primary Cultures of Epinephelus coioides

    PubMed Central

    Wang, Tao; Chen, Xiaoyan; Long, Xiaohua; Liu, Zhaopu; Yan, Shaohua

    2016-01-01

    Copper nanoparticles (Cu-NPs) were widely used in various industrial and commercial applications. The aim of this study was to analyze the cytotoxicity of Cu-NPs on primary hepatocytes of E.coioides compared with copper sulphate (CuSO4). Cultured cells were exposed to 0 or 2.4 mg Cu L-1 as CuSO4or Cu-NPs for 24-h. Results showed either form of Cu caused a dramatic loss in cell viability, more so in the CuSO4 than Cu-NPs treatment. Compared to control, either CuSO4 or Cu-NPs significantly increased reactive oxygen species(ROS) and malondialdehyde(MDA) concentration in hepatocytes by overwhelming total superoxide dismutase (T-SOD) activity, catalase(CAT) activity and glutathione(GSH) concentration. In addition, the antioxidative-related genes [SOD (Cu/Zn), SOD (Mn), CAT, GPx4] were also down-regulated. The apoptosis and necrosis percentage was significantly higher after the CuSO4 or Cu-NPs treatment than the control. The apoptosis was induced by the increased cytochrome c concentration in the cytosol and elevated caspase-3, caspase-8 and caspase-9 activities. Additionally, the apoptosis-related genes (p53, p38β and TNF-α) and protein (p53 protein) were up-regulated after the CuSO4 or Cu-NPs treatment, with CuSO4 exposure having a greater effect than Cu-NPs. In conclusion, Cu-NPs had similar types of toxic effects as CuSO4 on primary hepatocytes of E.coioides, but toxicity of CuSO4 was more severe than that of Cu-NPs. PMID:26890000

  8. Copper Nanoparticles and Copper Sulphate Induced Cytotoxicity in Hepatocyte Primary Cultures of Epinephelus coioides.

    PubMed

    Wang, Tao; Chen, Xiaoyan; Long, Xiaohua; Liu, Zhaopu; Yan, Shaohua

    2016-01-01

    Copper nanoparticles (Cu-NPs) were widely used in various industrial and commercial applications. The aim of this study was to analyze the cytotoxicity of Cu-NPs on primary hepatocytes of E.coioides compared with copper sulphate (CuSO4). Cultured cells were exposed to 0 or 2.4 mg Cu L-1 as CuSO4or Cu-NPs for 24-h. Results showed either form of Cu caused a dramatic loss in cell viability, more so in the CuSO4 than Cu-NPs treatment. Compared to control, either CuSO4 or Cu-NPs significantly increased reactive oxygen species(ROS) and malondialdehyde(MDA) concentration in hepatocytes by overwhelming total superoxide dismutase (T-SOD) activity, catalase(CAT) activity and glutathione(GSH) concentration. In addition, the antioxidative-related genes [SOD (Cu/Zn), SOD (Mn), CAT, GPx4] were also down-regulated. The apoptosis and necrosis percentage was significantly higher after the CuSO4 or Cu-NPs treatment than the control. The apoptosis was induced by the increased cytochrome c concentration in the cytosol and elevated caspase-3, caspase-8 and caspase-9 activities. Additionally, the apoptosis-related genes (p53, p38β and TNF-α) and protein (p53 protein) were up-regulated after the CuSO4 or Cu-NPs treatment, with CuSO4 exposure having a greater effect than Cu-NPs. In conclusion, Cu-NPs had similar types of toxic effects as CuSO4 on primary hepatocytes of E.coioides, but toxicity of CuSO4 was more severe than that of Cu-NPs.

  9. Intracellular degradation of chemically functionalized carbon nanotubes using a long-term primary microglial culture model.

    PubMed

    Bussy, Cyrill; Hadad, Caroline; Prato, Maurizio; Bianco, Alberto; Kostarelos, Kostas

    2016-01-07

    Chemically functionalized carbon nanotubes (f-CNTs) have been used in proof-of-concept studies to alleviate debilitating neurological conditions. Previous in vivo observations in brain tissue have suggested that microglia - acting as resident macrophages of the brain - play a critical role in the internalization of f-CNTs and their partial in situ biodegradation following a stereotactic administration in the cortex. At the same time, several reports have indicated that immune cells such as neutrophils, eosinophils and even macrophages could participate in the processing of carbon nanomaterials via oxidation processes leading to degradation, with surface properties acting as modulators of CNT biodegradability. In this study we questioned whether degradability of f-CNTs within microglia could be modulated depending on the type of surface functionalization used. We investigated the kinetics of degradation of multi-walled carbon nanotubes (MWNTs) functionalized via different chemical strategies that were internalized within isolated primary microglia over three months. A cellular model of rat primary microglia that can be maintained in cell culture for a long period of time was first developed. The Raman structural signature of the internalized f-CNTs was then studied directly in cells over a period of up to three months, following a single exposure to a non-cytotoxic concentration of three different f-CNTs (carboxylated, aminated and both carboxylated and aminated). Structural modifications suggesting partial but continuous degradation were observed for all nanotubes irrespective of their surface functionalization. Carboxylation was shown to promote more pronounced structural changes inside microglia over the first two weeks of the study.

  10. Cytotoxic mechanisms of hydrosulfide anion and cyanide anion in primary rat hepatocyte cultures.

    PubMed

    Thompson, Rodney W; Valentine, Holly L; Valentine, William M

    2003-06-30

    Hydrogen sulfide and hydrogen cyanide are known to compromise mitochondrial respiration through inhibition of cytochrome c oxidase and this is generally considered to be their primary mechanism of toxicity. Experimental studies and the efficiency of current treatment protocols suggest that H(2)S may exert adverse physiological effects through additional mechanisms. To evaluate the role of alternative mechanisms in H(2)S toxicity, the relative contributions of electron transport inhibition, uncoupling of mitochondrial respiration, and opening of the mitochondrial permeability transition pore (MPTP) to hydrosulfide and cyanide anion cytotoxicity in primary hepatocyte cultures were examined. Supplementation of hepatocytes with the glycolytic substrate, fructose, rescued hepatocytes from cyanide anion induced toxicity, whereas fructose supplementation increased hydrosulfide anion toxicity suggesting that hydrosulfide anion may compromise glycolysis in hepatocytes. Although inhibitors of the MPTP opening were protective for hydrosulfide anion, they had no effect on cyanide anion toxicity, consistent with an involvement of the permeability transition pore in hydrosulfide anion toxicity but not cyanide anion toxicity. Exposure of isolated rat liver mitochondria to hydrosulfide did not result in large amplitude swelling suggesting that if H(2)S induces the permeability transition it does so indirectly through a mechanism requiring other cellular components. Hydrosulfide anion did not appear to be an uncoupler of mitochondrial respiration in hepatocytes based upon the inability of oligomycin and fructose to protect hepatocytes from hydrosulfide anion toxicity. These findings support mechanisms additional to inhibition of cytochrome c oxidase in hydrogen sulfide toxicity. Further investigations are required to assess the role of the permeability transition in H(2)S toxicity, determine whether similar affects occur in other cell types or in vivo and evaluate whether this may

  11. Cytotoxicity Study on Luminescent Nanocrystals Containing Phospholipid Micelles in Primary Cultures of Rat Astrocytes

    PubMed Central

    Valente, Gianpiero; Fanizza, Elisabetta; Laquintana, Valentino; Denora, Nunzio; Fasano, Anna; Striccoli, Marinella; Colella, Matilde; Agostiano, Angela; Curri, M. Lucia; Liuzzi, Grazia Maria

    2016-01-01

    Luminescent colloidal nanocrystals (NCs) are emerging as a new tool in neuroscience field, representing superior optical probes for cellular imaging and medical diagnosis of neurological disorders with respect to organic fluorophores. However, only a limited number of studies have, so far, explored NC applications in primary neurons, glia and related cells. Indeed astrocytes, as resident cells in the central nervous system (CNS), play an important pathogenic role in several neurodegenerative and neuroinflammatory diseases, therefore enhanced imaging tools for their thorough investigation are strongly amenable. Here, a comprehensive and systematic study on the in vitro toxicological effect of core-shell type luminescent CdSe@ZnS NCs incorporated in polyethylene glycol (PEG) terminated phospholipid micelles on primary cultures of rat astrocytes was carried out. Cytotoxicity response of empty micelles based on PEG modified phospholipids was compared to that of their NC containing counterpart, in order to investigate the effect on cell viability of both inorganic NCs and micelles protecting NC surface. Furthermore, since the surface charge and chemistry influence cell interaction and toxicity, effect of two different functional groups terminating PEG-modified phospholipid micelles, namely amine and carboxyl group, respectively, was evaluated against bare micelles, showing that carboxyl group was less toxic. The ability of PEG-lipid micelles to be internalized into the cells was qualitatively and quantitatively assessed by fluorescence microscopy and photoluminescence (PL) assay. The results of the experiments clearly demonstrate that, once incorporated into the micelles, a low, not toxic, concentration of NCs is sufficient to be distinctly detected within cells. The overall study provides essential indications to define the optimal experimental conditions to effectively and profitably use the proposed luminescent colloidal NCs as optical probe for future in vivo

  12. Cytotoxicity Study on Luminescent Nanocrystals Containing Phospholipid Micelles in Primary Cultures of Rat Astrocytes.

    PubMed

    Latronico, Tiziana; Depalo, Nicoletta; Valente, Gianpiero; Fanizza, Elisabetta; Laquintana, Valentino; Denora, Nunzio; Fasano, Anna; Striccoli, Marinella; Colella, Matilde; Agostiano, Angela; Curri, M Lucia; Liuzzi, Grazia Maria

    2016-01-01

    Luminescent colloidal nanocrystals (NCs) are emerging as a new tool in neuroscience field, representing superior optical probes for cellular imaging and medical diagnosis of neurological disorders with respect to organic fluorophores. However, only a limited number of studies have, so far, explored NC applications in primary neurons, glia and related cells. Indeed astrocytes, as resident cells in the central nervous system (CNS), play an important pathogenic role in several neurodegenerative and neuroinflammatory diseases, therefore enhanced imaging tools for their thorough investigation are strongly amenable. Here, a comprehensive and systematic study on the in vitro toxicological effect of core-shell type luminescent CdSe@ZnS NCs incorporated in polyethylene glycol (PEG) terminated phospholipid micelles on primary cultures of rat astrocytes was carried out. Cytotoxicity response of empty micelles based on PEG modified phospholipids was compared to that of their NC containing counterpart, in order to investigate the effect on cell viability of both inorganic NCs and micelles protecting NC surface. Furthermore, since the surface charge and chemistry influence cell interaction and toxicity, effect of two different functional groups terminating PEG-modified phospholipid micelles, namely amine and carboxyl group, respectively, was evaluated against bare micelles, showing that carboxyl group was less toxic. The ability of PEG-lipid micelles to be internalized into the cells was qualitatively and quantitatively assessed by fluorescence microscopy and photoluminescence (PL) assay. The results of the experiments clearly demonstrate that, once incorporated into the micelles, a low, not toxic, concentration of NCs is sufficient to be distinctly detected within cells. The overall study provides essential indications to define the optimal experimental conditions to effectively and profitably use the proposed luminescent colloidal NCs as optical probe for future in vivo

  13. The difficult-to-cultivate coxsackieviruses A can productively multiply in primary culture of mouse skeletal muscle.

    PubMed

    Nsaibia, Siwar; Wagner, Stéphanie; Rondé, Philippe; Warter, Jean-Marie; Poindron, Philippe; Aouni, Mahjoub; Dorchies, Olivier M

    2007-01-01

    Coxsackieviruses A (CVA) are associated with several clinical manifestations such as aseptic meningitis and paralytic syndromes in humans. Most CVA are difficult-to-cultivate, which impedes their propagation and isolation from clinical material. Here, we tested the ability of cultivable (CVA-13, CVA-14), and difficult-to-cultivate (CVA-6, CVA-22) strains to infect primary cultures of skeletal muscle cells established from newborn mice. We found that such cultures sustained the multiplication of these CVA, as evidenced by the development of a cytopathic effect, already in the initial preparation or after passaging once. Cultures established for no more than 24h were sensitive to infection whereas older preparations were resistant. Using confocal microscopy after double-immunolabeling of the VP1 capsid protein and the muscle cell marker myosin, we demonstrated that only the myoblasts were infected, resulting in VP1 expression throughout their cytoplasm. Inoculation of infected cultures to suckling mice resulted in paralysis indicating that infection was productive. The nature of candidate receptors for virus entry in such cultures and the influence of cell culture conditions on the expression of these putative receptors are discussed. This work suggests that primary cultures of skeletal muscle cells could be used to propagate and isolate any CVA strain.

  14. Functional differentiation and alveolar morphogenesis of primary mammary cultures on reconstituted basement membrane

    SciTech Connect

    BARCELLOS-HOFF, M. H; AGGELER, J.; RAM, T. G; BISSELL, M. J

    1989-02-01

    An essential feature of mammary gland differentiation during pregnancy is the formation of alveoli composed of polarized epithelial cells, which, under the influence of lactogenic hormones, secrete vectorially and sequester milk proteins. Previous culture studies have described either organization of cells polarized towards lumina containing little or no demonstrable tissue-specific protein, or establishment of functional secretory cells exhibiting little or no glandular architecture. In this paper, we report that tissue-specific vectorial secretion coincides with the formation of functional alveoli-like structures by primary mammary epithelial cells cultured on a reconstituted basement membrane matrix (derived from Engelbreth-Holm-Swarm murine tumour). Morphogenesis of these unique three-dimensional structures was initiated by cell-directed remodelling of the exogenous matrix leading to reorganization of cells into matrixensheathed aggregates by 24 h after plating. The aggregates subsequently cavitated, so that by day 6 the cells were organized into hollow spheres in which apical cell surfaces faced lumina sealed by tight junctions and basal surfaces were surrounded by a distinct basal lamina. The profiles of proteins secreted into the apical (luminal) and basal (medium) compartments indicated that these alveoli-like structures were capable of an appreciable amount of vectorial secretion. Immunoprecipitation with a broad spectrum milk antiserum showed that more than 80% of caseins were secreted into the lumina, whereas iron-binding proteins (both lactoferrin and transferrin) were present in comparable amounts in each compartment. Thus, these mammary cells established protein targeting pathways directing milk-specific proteins to the luminal compartment. A time course monitoring secretory activity demonstrated that establishment of tissue-specific vectorial secretion and increased total and milk protein secretion coincided with functional alveolar

  15. The use of Fluoro-Jade in primary neuronal cell cultures.

    PubMed

    Schmuck, Gabriele; Kahl, Regine

    2009-04-01

    Fluoro-Jade (FJ) and its derivatives are widely used for histological staining of neurons undergoing neurodegeneration. With this dye, the entire structure of these neurons can be stained in a fast and reliable way in histopathological slices of the brain, with results comparable to those obtained with other methods such as the Nissle technique or silver staining. The question arose as to whether this method might be useful for in vitro neuronal cell cultures. Primary cortical neuronal cell cultures have been used as a sensitive and reliable system to detect compounds which induce neurodegenerative lesions (Schmuck et al. 2000). Additionally, various biochemical endpoints in this system allow the mode of action of these compounds to be identified. The target mechanism of FJ staining is unknown, and it may therefore be useful to compare FJ staining with one of the central endpoints in compound-induced neurodegeneration, interaction with the cytoskeleton as demonstrated by accumulations of neurofilaments (200 kD). Cortical neuronal cells were cultivated under standardized serum-free conditions. Once they had developed a stable network, the cells were treated with acrylamide, mipafox, diethyldithiocarbamate, glutamate, paraquat, paraoxon, and IDPN (ss,ss-imino dipropionitrile) for 7 days in the concentration range of 0.1-50 microg/ml. One half of the cell culture samples were tested directly after 7 days, the others were allowed to recover during a 7-day treatment-free period. Subsequently viability testing and quantification for FJ staining were performed. All compounds except paraoxon increased FJ staining after 7 days, and this signal increased slightly during the recovery period with glutamate and acrylamide. With mipafox and IDPN the signal decreased slightly. Paraoxon increased FJ staining only after the recovery period. The intensity of FJ staining did not always correlate with neurofilament destruction or cytotoxicity. It can therefore be assumed that FJ

  16. Human Peripheral Blood Mononuclear Cells Cultured in Normal and Hyperglycemic Media in Simulated Microgravity Using NASA Bioreactors

    NASA Technical Reports Server (NTRS)

    Lawless, DeSales

    2003-01-01

    We sought answers to several questions this summer at NASA Johnson Space Center. Initial studies involved the in vitro culture of human peripheral blood mononuclear in cells in different conditioned culture media. Several human cancer clones were similarly studied to determine responses to aberrant glycosylation by the argon laser. The cells were grown at unit gravity in flasks and in simulated microgravity using NASA bioreactors. The cells in each instance were analyzed by flow cytometry. Cell cycle analysis was acquired by staining nuclear DNA with propidium iodide. Responses to the laser stimulation was measured by observing autofluorescence emitted in the green and red spectra after stimulation. Extent of glycosylation correlated with the intensity of the laser stimulated auto-fluorescence. Our particular study was to detect and monitor aberrant glycosylation and its role in etiopathogenesis. Comparisons were made between cells known to be neoplastic and normal cell controls using the same Laser Induced Autofluorescence technique. Studies were begun after extensive literature searches on using the antigen presenting potential of dendritic cells to induce proliferation of antigen specific cytotoxic T-cells. The Sendai virus served as the antigen. Our goal is to generate sufficient numbers of such cells in the simulated microgravity environment for use in autologous transplants of virally infected individuals including those positive for hepatitis and HIV.

  17. Analysis on the Relationship between Trust Culture and Prejudices in Primary Schools

    ERIC Educational Resources Information Center

    Erdogan, Cetin

    2016-01-01

    Problem Statement: Trust is crucial for creating a positive culture in the school environment, which is called as trust culture. On the other hand, prejudice is thought to be a potential barrier for creating trust culture in schools. Thus, it is meaningful to examine the relationship between trust culture and prejudice in schools and then to…

  18. Culturally Responsive Literacy Instruction: An Investigation of Primary Grade Teachers' Attitudes, Beliefs and Practices with African American Students

    ERIC Educational Resources Information Center

    Reid-Agren, Kathleen J.

    2010-01-01

    The purpose of this study was to investigate primary grade teachers' attitudes, beliefs and practices concerning Culturally Responsive Literacy Instruction (CRLI) with African American students. Through a mixed methods research design, quantitative and qualitative data sources were collected and analyzed sequentially. The participant school…

  19. The Sustainable Impact of a Short Comparative Teaching Placement Abroad on Primary School Language Teachers' Professional, Linguistic and Cultural Skills

    ERIC Educational Resources Information Center

    Driscoll, Patricia; Rowe, Janet Elizabeth; Thomae, Manuela

    2014-01-01

    Recent research shows an increase in the number of trained teachers teaching foreign languages to learners aged 7-11 in English primary schools. Part of this increase stems from a government-funded four-week teaching placement abroad as part of a languages programme in initial teacher education (ITE). Student teachers' cultural, linguistic and…

  20. The Implementation of a Social Constructivist Approach in Primary Science Education in Confucian Heritage Culture: The Case of Vietnam

    ERIC Educational Resources Information Center

    H?ng, Ngô Vu Thu; Meijer, Marijn Roland; Bulte, Astrid M. W.; Pilot, Albert

    2015-01-01

    Social constructivism has been increasingly studied and implemented in science school education. Nevertheless, there is a lack of holistic studies on the implementation of social constructivist approach in primary science education in Confucian heritage culture. This study aims to determine to what extent a social constructivist approach is…

  1. Increased formation of autophagosomes in ectromelia virus-infected primary culture of murine bone marrow-derived macrophages.

    PubMed

    Martyniszyn, L; Szulc-Dąbrowska, L; Boratyńska-Jasińska, A; Niemiałtowski, M

    2013-01-01

    Induction of autophagy by ectromelia virus (ECTV) in primary cultures of bone marrow-derived macrophages (BMDMs) was investigated. The results showed that ECTV infection of BMDMs resulted in increased formation of autophagosomes, increased level of LC3-II protein present in aggregates and extensive cytoplasmic vacuolization. These data indicate an increased autophagic activity in BMDMs during ECTV infection.

  2. Comparative growth, cross stress resistance, transcriptomics of Streptococcus pyogenes cultured under low shear modeled microgravity and normal gravity

    PubMed Central

    Kalpana, Duraisamy; Im, Chanki; Lee, Yang Soo

    2015-01-01

    Streptococcus pyogenes is commonly found on pharynx, mouth and rarely on skin, lower gastrointestinal tract. It is a potential pathogen causing tonsillitis, pneumonia, endocarditis. The present study was undertaken to study the effects of low shear modeled microgravity on growth, morphology, antibiotic resistance, cross-stress resistance to various stresses and alteration in gene expression of S. pyogenes. The growth analysis performed using UV–Visible spectroscopy indicated decrease in growth of S. pyogenes under low shear modeled microgravity. Morphological analysis by Bio-transmission electron microscopy (TEM), Bio-scanning electron microscopy (SEM) did not reveal much difference between normal and low shear modeled microgravity grown S. pyogenes. The sensitivity of S. pyogenes to antibiotics ampicillin, penicillin, streptomycin, kanamycin, hygromycin, rifampicin indicates that the bacterium is resistant to hygromycin. Further S. pyogenes cultured under low shear modeled microgravity was found to be more sensitive to ampicillin and rifampicin as compared with normal gravity grown S. pyogenes. The bacteria were tested for the acid, osmotic, temperature and oxidative cross stress resistances. The gene expression of S. pyogenes under low shear modeled microgravity analyzed by microarray revealed upregulation of 26 genes and down regulation of 22 genes by a fold change of 1.5. PMID:26858535

  3. Comparative growth, cross stress resistance, transcriptomics of Streptococcus pyogenes cultured under low shear modeled microgravity and normal gravity.

    PubMed

    Kalpana, Duraisamy; Im, Chanki; Lee, Yang Soo

    2016-01-01

    Streptococcus pyogenes is commonly found on pharynx, mouth and rarely on skin, lower gastrointestinal tract. It is a potential pathogen causing tonsillitis, pneumonia, endocarditis. The present study was undertaken to study the effects of low shear modeled microgravity on growth, morphology, antibiotic resistance, cross-stress resistance to various stresses and alteration in gene expression of S. pyogenes. The growth analysis performed using UV-Visible spectroscopy indicated decrease in growth of S. pyogenes under low shear modeled microgravity. Morphological analysis by Bio-transmission electron microscopy (TEM), Bio-scanning electron microscopy (SEM) did not reveal much difference between normal and low shear modeled microgravity grown S. pyogenes. The sensitivity of S. pyogenes to antibiotics ampicillin, penicillin, streptomycin, kanamycin, hygromycin, rifampicin indicates that the bacterium is resistant to hygromycin. Further S. pyogenes cultured under low shear modeled microgravity was found to be more sensitive to ampicillin and rifampicin as compared with normal gravity grown S. pyogenes. The bacteria were tested for the acid, osmotic, temperature and oxidative cross stress resistances. The gene expression of S. pyogenes under low shear modeled microgravity analyzed by microarray revealed upregulation of 26 genes and down regulation of 22 genes by a fold change of 1.5.

  4. IL-6 Augments Angiotensinogen in Primary Cultured Renal Proximal Tubular Cells

    PubMed Central

    Satou, Ryousuke; Gonzalez-Villalobos, Romer A.; Miyata, Kayoko; Ohashi, Naro; Urushihara, Maki; Acres, Omar W.; Navar, L. Gabriel; Kobori, Hiroyuki

    2009-01-01

    In human kidneys, the mechanisms underlying angiotensinogen (AGT) augmentation by interleukin 6 (IL-6) are poorly understood and the only information available is in HK-2, immortalized human renal proximal tubular epithelial cells. Therefore, the present study was performed to elucidate the effects of IL-6 on AGT expression in primary cultured human renal proximal tubular epithelial cells (RPTEC) after characterization of HK-2 and RPTEC. RPTEC showed low basal AGT mRNA (11±1%) and protein (7.0±0.9%) expression, high IL-6 receptor (IL-6R) expression (282±17%), and low basal NF-κB (43±7%) and STAT3 (43±7%) activities compared to those in HK-2. In RPTEC, AGT mRNA and protein expressions were enhanced by IL-6 (172±31% and 378±39%, respectively). This AGT augmentation was attenuated by an IL-6R antibody. STAT3 phosphorylation (366±55% at 30 min) and translocation were enhanced by IL-6. The AGT augmentation was attenuated by a STAT3 inhibitor. These data indicate that IL-6 increases AGT expression via STAT3 pathway in RPTEC. PMID:19583994

  5. Comparison of rat and hamster hepatocyte primary culture/DNA repair assays

    SciTech Connect

    Kornbrust, D.J.; Barfknect, T.R.

    1984-01-01

    Previous studies have demonstrated marked differences in the capacity of hepatocytes from rats or hamsters to mediate the metabolic activation of chemical carcinogens to genotoxic (i.e., mutagenic) products. Thus far, very few investigations of species differences in DNA repair have been performed. Therefore, a comparison of the relative extent of DNA repair elicited by various genotoxic chemicals in rat and hamster hepatocyes was conducted, using the hepatocyte primary culture/DNA repair (HPC/DR) assay. Of the ll chemicals tested, eight were more potent in inducing DNA repair in hamster hepatocytes than in rat hepatocytes. Dimethylnitrosamine, diethylnitrosamine, 2-acetylaminofluorene, 9-aminoacridine, pararosaniline hydrochloride, 1-naphthylamine, benzidine and 1,2,3,4-diepoxybutane were all active in hamster hepatocytes at a concentration at least ten times less than the lowest effective concentration in rat hepatocytes. The direct-acting alkylating agent, methylmethane sulfonate, was equipotent inducing DNA repair in both rat and hamster hepatocytes, indicating that the differences in DNA repair observed for the other chemicals were probably not a result of species differences in DNA repair capacities. In contrast, 1-nitropyrene produced a greater DNA repair response in rat hepatocyes than hamster hepatocytes, while the bacterial mutagen 3-(chloromethyl)pyridine hydrochloride was inactive in both hepatocyte systems. These studies demonstrate the feasibility of using hamster hepatocytes in the HPC/DR assay and illustrate the utility of performing the assay with hepatocytes from more than one species.

  6. Differences in morphogenesis of 3D cultured primary human osteoblasts under static and microfluidic growth conditions.

    PubMed

    Altmann, Brigitte; Löchner, Anne; Swain, Michael; Kohal, Ralf-Joachim; Giselbrecht, Stefan; Gottwald, Eric; Steinberg, Thorsten; Tomakidi, Pascal

    2014-03-01

    As information on osteoblast mechanosensitivity response to biomechanical cues in three-dimensional (3D) in vitro microenvironments is sparse, the present study compared morphogenesis of primary human alveolar bone osteoblasts (PHABO) under microchip-based 3D-static conditions, and 3D-fluid flow-mediated biomechanical stimulation in perfusion bioreactors. Discrimination of the respective microenvironment by differential morphogenesis was evident from fluid flow-induced PHABO reorganization into rotund bony microtissue, comprising more densely packed multicellular 3D-aggregates, while viability of microtissues was flow rate dependent. Time-lapse microscopy and simple modeling of biomechanical conditions revealed that physiologically relevant fluid flow-mediated PHABO stimulation was associated with formation of mulberry-like PHABO aggregates within the first 24 h. Differential extracellular matrix deposition patterns and gene expression modulation in PHABO aggregates at day 7 further indicates progressive osteoblast differentiation exclusively in perfusion culture-developed bony microtissues. The results of our study strongly suggest PHABO morphogenesis as discriminator of microenvironmental growth conditions, which in case of the microfluidic 3D microchip-bioreactor are substantiated by triggering in vitro bone microtissue formation concomitant with progressive osteoblastic differentiation. Such microtissue outcomes provide unique insight for mechanobiological studies in response to biomechanical fluid flow cues, and clinically appear promising for in vitro PHABO preconditioning, enabling innovative bone augmentation procedures.

  7. Gene expression in primary cultured astrocytes affected by aluminum: alteration of chaperons involved in protein folding

    PubMed Central

    Aremu, David A.; Ezomo, Ojeiru F.

    2010-01-01

    Objectives Aluminum is notorious as a neurotoxic metal. The aim of our study was to determine whether endoplasmic reticulum (ER) stress is involved in aluminum-induced apoptosis in astrocytes. Methods Mitochondrial RNA (mRNA) was analyzed by reverse transcription (RT)-PCR following pulse exposure of aluminum glycinate to primary cultured astrocytes. Tunicamycin was used as a positive control. Results Gene expression analysis revealed that Ire1β was up-regulated in astrocytes exposed to aluminum while Ire1α was up-regulated by tunicamycin. Exposure to aluminum glycinate, in contrast to tunicamycin, seemed to down-regulate mRNA expression of many genes, including the ER resident molecular chaperone BiP/Grp78 and Ca2+-binding chaperones (calnexin and calreticulin), as well as stanniocalcin 2 and OASIS. The down-regulation or non-activation of the molecular chaperons, whose expressions are known to be protective by increasing protein folding, may spell doom for the adaptive response. Exposure to aluminum did not have any significant effects on the expression of Bax and Bcl2 in astrocytes. Conclusions The results of this study demonstrate that aluminum may induce apoptosis in astrocytes via ER stress by impairing the protein-folding machinery. PMID:21432213

  8. Nogo receptor 1 is expressed in both primary cultured glial cells and neurons

    PubMed Central

    Ukai, Junichi; Imagama, Shiro; Ohgomori, Tomohiro; Ito, Zenya; Ando, Kei; Ishiguro, Naoki; Kadomatsu, Kenji

    2016-01-01

    ABSTRACT Nogo receptor (NgR) is common in myelin-derived molecules, i.e., Nogo, MAG, and OMgp, and plays important roles in both axon fasciculation and the inhibition of axonal regeneration. In contrast to NgR’s roles in neurons, its roles in glial cells have been poorly explored. Here, we found a dynamic regulation of NgR1 expression during development and neuronal injury. NgR1 mRNA was consistently expressed in the brain from embryonic day 18 to postnatal day 25. In contrast, its expression significantly decreased in the spinal cord during development. Primary cultured neurons, microglia, and astrocytes expressed NgR1. Interestingly, a contusion injury in the spinal cord led to elevated NgR1 mRNA expression at the injury site, but not in the motor cortex, 14 days after injury. Consistent with this, astrocyte activation by TGFβ1 increased NgR1 expression, while microglia activation rather decreased NgR1 expression. These results collectively suggest that NgR1 expression is enhanced in a milieu of neural injury. Our findings may provide insight into the roles of NgR1 in glial cells. PMID:27578914

  9. Cultural responses to pain in UK children of primary school age: a mixed-methods study.

    PubMed

    Azize, Pary M; Endacott, Ruth; Cattani, Allegra; Humphreys, Ann

    2014-06-01

    Pain-measurement tools are often criticized for not addressing the influence of culture and ethnicity on pain. This study examined how children who speak English as a primary or additional language discuss pain. Two methods were used in six focus group interviews with 34 children aged 4-7 years: (i) use of drawings from the Pediatric Pain Inventory to capture the language used by children to describe pain; and (ii) observation of the children's placing of pain drawings on red/amber/green paper to denote perceived severity of pain. The findings demonstrated that children with English as an additional language used less elaborate language when talking about pain, but tended to talk about the pictures prior to deciding where they should be placed. For these children, there was a positive significant relationship between language, age, and length of stay in the UK. The children's placement of pain drawings varied according to language background, sex, and age. The findings emphasize the need for sufficient time to assess pain adequately in children who do not speak English as a first language.

  10. Ambroxol inhibits rhinovirus infection in primary cultures of human tracheal epithelial cells.

    PubMed

    Yamaya, Mutsuo; Nishimura, Hidekazu; Nadine, Lusamba Kalonji; Ota, Chiharu; Kubo, Hiroshi; Nagatomi, Ryoichi

    2014-04-01

    The mucolytic drug ambroxol hydrochloride reduces the production of pro-inflammatory cytokines and the frequency of exacerbation in patients with chronic obstructive pulmonary disease (COPD). However, the inhibitory effects of ambroxol on rhinovirus infection, the major cause of COPD exacerbations, have not been studied. We examined the effects of ambroxol on type 14 rhinovirus (RV14) infection, a major RV group, in primary cultures of human tracheal epithelial cells. RV14 infection increased virus titers and cytokine content in the supernatants and RV14 RNA in the cells. Ambroxol (100 nM) reduced RV14 titers and cytokine concentrations of interleukin (IL)-1β, IL-6 and IL-8 in the supernatants and RV14 RNA in the cells after RV14 infection, in addition to reducing susceptibility to RV14 infection. Ambroxol also reduced the expression of intercellular adhesion molecule-1 (ICAM-1), the receptor for RV14, and the number of acidic endosomes from which RV14 RNA enters the cytoplasm. In addition, ambroxol reduced the activation of the transcription factor nuclear factor kappa B (NF-κB) in the nucleus. These results suggest that ambroxol inhibits RV14 infection partly by reducing ICAM-1 and acidic endosomes via the inhibition of NF-κB activation. Ambroxol may modulate airway inflammation by reducing the production of cytokines in rhinovirus infection.

  11. Ganglioside inhibition of glutamate-mediated protein kinase C translocation in primary cultures of cerebellar neurons

    SciTech Connect

    Vaccarino, F.; Guidotti, A.; Costa, E.

    1987-12-01

    In primary cultures of cerebellar granule cells, protein kinase C (PKC) translocation and activation can be triggered by the stimulation of excitatory amino acid neurotransmitter receptors. Glutamate evokes a dose-related translocation of 4-..beta..-(/sup 3/H)phorbol 12,13-dibutyrate /(/sup 3/H)-P(BtO)/sub 2// binding sites from the cytosol to the neuronal membrane and stimulates the incorporation of /sup 32/P into a number of membrane proteins, particularly protein bands in the range of 80, 50, and 40 kDa. The glutamate-evoked PKC translocation is Mg/sup 2 +/ sensitive, is prevented by 2-amino-5-phosphonovalerate and phencyclidine, is not inhibited by nitrendipine (a voltage-dependent Ca/sup 2 +/-channel-blocker) but is abolished by the removal of Ca/sup 2 +/ from the incubation medium, suggesting that glutamate-mediated Ca/sup 2 +/ influx is operative in the redistribution of PKC. Exposure of granule cells to the gangliosides trisialosylgangliotetraglycosylceramide (GT1b) of monosialosylgangliotetraglycosylceramide (GM1) inhibits the translocation and activation of PKC evoked by glutamate. These glycosphingolipids fail to interfere with glutamate binding to its high-affinity recognition site of with the (/sup 3/H)P(BtO)/sub 2/ binding, nor do they affect the Ca/sup 2 +/ influx. These gangliosides may prevent PKC translocation by interfering with the PKC binding to the neuronal membrane phosphatidylserine.

  12. Phosphatidylcholine resynthesis from components of internalized phospholipids in rat granular pneumocytes in primary culture

    SciTech Connect

    Chander, A.; Reicherter, J.; Fisher, A.B.

    1986-05-01

    Uptake, degradation and reutilization of surfactant phospholipids was investigated by incubating granular pneumocytes in primary culture with 0.2 mM liposomal phosphatidylcholine containing (/sup 3/H-methyl)choline labeled dipalmitoyl PC. Trypsin-resistant cell associated liposome radioactivity in PC declined steadily with time of incubation to 50% of total radioactivity by 140 min. In the water soluble fraction, most of the radioactivity was present in glycerophosphorylcholine which increased steadily to 13% of total cell associated radioactivity. While the proportion of radioactivity in choline remained unchanged, it increased with time in CDP-choline and phosphorylcholine suggesting reutilization of choline for PC resynthesis. In lamellar bodies isolated from these cells, less than 10% of PC label was present in unsaturated PC. In the microsomal fraction the label in unsaturated PC at 21 min was 56% of total PC which increased to 71% by 140 min of incubation with liposomes (slope = 0.19%/min; r = 0.67) suggesting metabolic reutilization of dipalmitoyl PC in this compartment. These observations indicate that granular pneumocytes degrade internalized PC and resynthesize PC de novo from degradation products.

  13. Anandamide, Acting via CB2 Receptors, Alleviates LPS-Induced Neuroinflammation in Rat Primary Microglial Cultures

    PubMed Central

    Malek, Natalia; Popiolek-Barczyk, Katarzyna; Mika, Joanna; Przewlocka, Barbara; Starowicz, Katarzyna

    2015-01-01

    Microglial activation is a polarized process divided into potentially neuroprotective phenotype M2 and neurotoxic phenotype M1, predominant during chronic neuroinflammation. Endocannabinoid system provides an attractive target to control the balance between microglial phenotypes. Anandamide as an immune modulator in the central nervous system acts via not only cannabinoid receptors (CB1 and CB2) but also other targets (e.g., GPR18/GPR55). We studied the effect of anandamide on lipopolysaccharide-induced changes in rat primary microglial cultures. Microglial activation was assessed based on nitric oxide (NO) production. Analysis of mRNA was conducted for M1 and M2 phenotype markers possibly affected by the treatment. Our results showed that lipopolysaccharide-induced NO release in microglia was significantly attenuated, with concomitant downregulation of M1 phenotypic markers, after pretreatment with anandamide. This effect was not sensitive to CB1 or GPR18/GPR55 antagonism. Administration of CB2 antagonist partially abolished the effects of anandamide on microglia. Interestingly, administration of a GPR18/GPR55 antagonist by itself suppressed NO release. In summary, we showed that the endocannabinoid system plays a crucial role in the management of neuroinflammation by dampening the activation of an M1 phenotype. This effect was primarily controlled by the CB2 receptor, although functional cross talk with GPR18/GPR55 may occur. PMID:26090232

  14. Modification of distinct ion channels differentially modulates Ca2+ dynamics in primary cultured rat ventricular cardiomyocytes

    PubMed Central

    Li, Xichun; Shen, Liping; Zhao, Fang; Zou, Xiaohan; He, Yuwei; Zhang, Fan; Zhang, Chunlei; Yu, Boyang; Cao, Zhengyu

    2017-01-01

    Primary cultured cardiomyocytes show spontaneous Ca2+ oscillations (SCOs) which not only govern contractile events, but undergo derangements that promote arrhythmogenesis through Ca2+ -dependent mechanism. We systematically examined influence on SCOs of an array of ion channel modifiers by recording intracellular Ca2+ dynamics in rat ventricular cardiomyocytes using Ca2+ specific fluorescence dye, Fluo-8/AM. Voltage-gated sodium channels (VGSCs) activation elongates SCO duration and reduces SCO frequency while inhibition of VGSCs decreases SCO frequency without affecting amplitude and duration. Inhibition of voltage-gated potassium channel increases SCO duration. Direct activation of L-type Ca2+ channels (LTCCs) induces SCO bursts while suppressing LTCCs decreases SCO amplitude and slightly increases SCO frequency. Activation of ryanodine receptors (RyRs) increases SCO duration and decreases both SCO amplitude and frequency while inhibiting RyRs decreases SCO frequency without affecting amplitude and duration. The potencies of these ion channel modifiers on SCO responses are generally consistent with their affinities in respective targets demonstrating that modification of distinct targets produces different SCO profiles. We further demonstrate that clinically-used drugs that produce Long-QT syndrome including cisapride, dofetilide, sotalol, and quinidine all induce SCO bursts while verapamil has no effect. Therefore, occurrence of SCO bursts may have a translational value to predict cardiotoxicants causing Long-QT syndrome. PMID:28102360

  15. PACAP27 regulates ciliary function in primary cultures of rat brain ependymal cells.

    PubMed

    Mönkkönen, K S; Mnkkönen, K S; Hirst, R A; Laitinen, J T; O'Callaghan, C

    2008-01-01

    Ependymal cells line the brain ventricles and separate the CSF from the underlying neuronal tissue. The function of ependymal cilia is largely unclear however they are reported to be involved in the regulation of CSF homeostasis and host defence against pathogens. Here we present data that implicates a role of pituitary adenylate cyclase-activating polypeptide (PACAP) in the inhibition of ependymal ciliary function, and also that the PACAP effects are not entirely dependent on adenylyl cyclase activation. Primary ependymal cultures were treated with increasing doses of PACAP27 or adenylyl cyclase toxin (ACT), and ciliary beating was recorded using high-speed digital video imaging. Ciliary beat frequency (CBF) and amplitude were determined from the videos. Ependymal CBF and ciliary amplitude were attenuated by PACAP27 in a concentration- and time-dependent manner. The peptide antagonist PACAP6-27 blocked PACAP27-induced decreases in amplitude and CBF. Treatment with ACT caused a decrease in amplitude but had no effect on CBF, this suggests that the inhibition of CBF and amplitude seen with PACAP27 may not be completely explained by G(s)-AC-cAMP pathway. We present here the first observational study to show that activation of PAC1 receptors with PACAP27 has an important role to play in the regulation of ependymal ciliary function.

  16. Neurogenic and neurotrophic effects of BDNF peptides in mouse hippocampal primary neuronal cell cultures.

    PubMed

    Cardenas-Aguayo, Maria del Carmen; Kazim, Syed Faraz; Grundke-Iqbal, Inge; Iqbal, Khalid

    2013-01-01

    The level of brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, is down regulated in Alzheimer's disease (AD), Parkinson's disease (PD), depression, stress, and anxiety; conversely the level of this neurotrophin is increased in autism spectrum disorders. Thus, modulating the level of BDNF can be a potential therapeutic approach for nervous system pathologies. In the present study, we designed five different tetra peptides (peptides B-1 to B-5) corresponding to different active regions of BDNF. These tetra peptides were found to be non-toxic, and they induced the expression of neuronal markers in mouse embryonic day 18 (E18) primary hippocampal neuronal cultures. Additionally, peptide B-5 induced the expression of BDNF and its receptor, TrkB, suggesting a positive feedback mechanism. The BDNF peptides induced only a moderate activation (phosphorylation at Tyr 706) of the TrkB receptor, which could be blocked by the Trk's inhibitor, K252a. Peptide B-3, when combined with BDNF, potentiated the survival effect of this neurotrophin on H(2)O(2)-treated E18 hippocampal cells. Peptides B-3 and B-5 were found to work as partial agonists and as partial antagonists competing with BDNF to activate the TrkB receptor in a dose-dependent manner. Taken together, these results suggest that the described BDNF tetra peptides are neurotrophic, can modulate BDNF signaling in a partial agonist/antagonist way, and offer a novel therapeutic approach to neural pathologies where BDNF levels are dysregulated.

  17. Astrocytes Enhance Streptococcus suis-Glial Cell Interaction in Primary Astrocyte-Microglial Cell Co-Cultures.

    PubMed

    Seele, Jana; Nau, Roland; Prajeeth, Chittappen K; Stangel, Martin; Valentin-Weigand, Peter; Seitz, Maren

    2016-06-13

    Streptococcus (S.) suis infections are the most common cause of meningitis in pigs. Moreover, S. suis is a zoonotic pathogen, which can lead to meningitis in humans, mainly in adults. We assume that glial cells may play a crucial role in host-pathogen interactions during S. suis infection of the central nervous system. Glial cells are considered to possess important functions during inflammation and injury of the brain in bacterial meningitis. In the present study, we established primary astrocyte-microglial cell co-cultures to investigate interactions of S. suis with glial cells. For this purpose, microglial cells and astrocytes were isolated from new-born mouse brains and characterized by flow cytometry, followed by the establishment of astrocyte and microglial cell mono-cultures as well as astrocyte-microglial cell co-cultures. In addition, we prepared microglial cell mono-cultures co-incubated with uninfected astrocyte mono-culture supernatants and astrocyte mono-cultures co-incubated with uninfected microglial cell mono-culture supernatants. After infection of the different cell cultures with S. suis, bacteria-cell association was mainly observed with microglial cells and most prominently with a non-encapsulated mutant of S. suis. A time-dependent induction of NO release was found only in the co-cultures and after co-incubation of microglial cells with uninfected supernatants of astrocyte mono-cultures mainly after infection with the capsular mutant. Only moderate cytotoxic effects were found in co-cultured glial cells after infection with S. suis. Taken together, astrocytes and astrocyte supernatants increased interaction of microglial cells with S. suis. Astrocyte-microglial cell co-cultures are suitable to study S. suis infections and bacteria-cell association as well as NO release by microglial cells was enhanced in the presence of astrocytes.

  18. PRIMARY CULTURE OF CHOROIDAL EPITHELIAL CELLS: CHARACTERIZATION OF AN IN VITRO MODEL OF BLOOD-CSF BARRIER

    PubMed Central

    ZHENG, WEI; ZHAO, QIUQU; GRAZIANO, JOSEPH H.

    2016-01-01

    Summary A primary rat choroidal epithelial cell culture system was developed to investigate mechanisms of heavy metal toxicity on the blood-cerebrospinal fluid (CSF) barrier. Epithelial cells were dissociated from choroidal tissue by pronase digestion and cultured in standard DMEM culture media supplemented with 10% fetal bovine serum and 10 ng epithelial growth factor per ml. The procedure yielded 2–5 × 104 cells from pooled plexuses of three to four rats, and a viability of 77–85%. The cultures displayed a dominant polygonal type of epithelial cells, with a population doubling time of 2–3 d. The cultures were of distinct choroidal epithelial origins. For example, immunocytochemical studies using monospecific rabbit anti-rat TTR polyclonal antibody revealed a strong positive stain of transthyretin (TTR), a thyroxine transport protein exclusively produced by the choroidal epithelia. Also, reverse-transcriptase polymerase chain reaction (PCR) confirmed the presence of specific TTR mRNA in the cultures. The cultures were further adapted to grow on a freely permeable membrane sandwiched between two culture chambers. The formation of an impermeable confluent monolayer occurred within 5 d after seeding and was verified by the presence of a steady electrical resistance across the membrane (80 ± 10 ohm per cm2). The epithelial barriers appeared to actively transport [125I]-thyroxine from the basal to apical chamber. These results suggest that this primary cell culture system possesses typical choroidal epithelial characteristics and appears to be a suitable model for in vitro mechanistic investigations of blood–CSF barrier. PMID:9542634

  19. Malachite green toxicity assessed on Asian catfish primary cultures of peripheral blood mononuclear cells by a proteomic analysis.

    PubMed

    Pierrard, Marie-Aline; Kestemont, Patrick; Delaive, Edouard; Dieu, Marc; Raes, Martine; Silvestre, Frédéric

    2012-06-15

    The potential genotoxic and carcinogenic properties reported for malachite green (MG) and the frequent detection of MG residues in fish and fish products, despite the ban of MG, have recently generated great concern. Additional toxicological data are required for a better understanding of the mechanism of action and a more comprehensive risk assessment for the exposure of fish to this fungicide. To date, the use of fish peripheral blood mononuclear cells (PBMCs) has not been exploited as a tool in the assessment of the toxicity of chemicals. However, PBMCs are exposed to toxicants and can be easily collected by blood sampling. The present study aims at better understanding the effects of MG by a proteomic analysis of primary cultured PBMC from the Asian catfish, Pangasianodon hypophthalmus, exposed to MG. The two lowest concentrations of 1 and 10 ppb were selected based on the MTS (water soluble tetrazolium salts) cytotoxicity test. Using a proteomic analysis (2D-DIGE), we showed that 109 proteins displayed significant changes in abundance in PBMC exposed during 48 h to MG. Most of these proteins were successfully identified by nano LC-MS/MS and validated through the Peptide and Protein Prophet of Scaffold™ software, but only 19 different proteins were considered corresponding to a single identification per spot. Our data suggest that low concentrations of MG could affect the mitochondrial metabolic functions, impair some signal transduction cascades and normal cell division, stimulate DNA repair and disorganize the cytoskeleton. Altogether, these results confirm that the mitochondrion is a target of MG toxicity. Further studies on the identified proteins are needed to better understand the mechanisms of MG toxicity in fish produced for human consumption.

  20. Amenorrhea - primary

    MedlinePlus

    ... of periods - primary Images Primary amenorrhea Normal uterine anatomy (cut section) Absence of menstruation (amenorrhea) References Bulun SE. The physiology and pathology of the female reproductive axis. In: ...

  1. Toxicity of green tea extracts and their constituents in rat hepatocytes in primary culture.

    PubMed

    Schmidt, M; Schmitz, H-J; Baumgart, A; Guédon, D; Netsch, M I; Kreuter, M-H; Schmidlin, C B; Schrenk, D

    2005-02-01

    Recent reports on sporadic cases of liver disorders (acute hepatitis, icterus, hepatocellular necrosis) after ingestion of dietary supplements based on hydro-alcoholic extracts from green tea leaves led to restrictions of the marketing of such products in certain countries of the EU. Since green tea is considered to exert a number of beneficial health effects, and, therefore, green tea products are widely used as dietary supplements, we were interested in the possible mechanism of hepatotoxicity of green tea extracts and in the components involved in such effects. Seven hours after seeding on collagen, rat hepatocytes in primary culture were treated with various hydro-alcoholic green tea extracts (two different native 80% ethanolic dry extracts and an 80% ethanolic dry extract cleared from lipophilic compounds). Cells were washed, and reduction of resazurin, used as a viability parameter monitoring intact mitochondrial function, was determined. It was found that all seven green tea extracts examined enhanced resazurin reduction significantly at a concentration range of 100-500 microg/ml medium, while a significant decrease was observed at 1-3mg/ml medium. Decreased levels were concomitant with abundant necrosis as observed by microscopic inspection of the cultures and with increased leakage of lactate dehydrogenase activity from the cells. In a separate series of experiments, the green tea constituents (-)-epicatechin, (-)-epigallocatechin-3-gallate, caffeine and theanine were tested at concentrations reflecting their levels in a typical green tea extract. Synthetic (+)-epigallocatechin (200 microM) was used for comparison. Cytotoxicity was found with (-)-epigallocatechin-3-gallate only. The concomitant addition of 0.25 mM ascorbate/0.05 mM alpha-tocopherol had no influence on cytotoxicity. In conclusion, our results suggest that high concentrations of green tea extract can exert acute toxicity in rat liver cells. (-)-Epigallocatechin-3-gallate seems to be a key

  2. Functional activity of the two promoters of the myosin alkali light chain gene in primary muscle cell cultures: comparison with other muscle gene promoters and other culture systems.

    PubMed Central

    Daubas, P; Klarsfeld, A; Garner, I; Pinset, C; Cox, R; Buckingham, M

    1988-01-01

    Proximal upstream flanking sequences of the mouse myosin alkali light chain gene encoding MLC1F and MLC3F, the mouse alpha-cardiac actin gene and the chicken gene for the alpha-subunit of the acetylcholine receptor were linked to the bacterial chloramphenicol acetyl transferase (CAT) gene and transfected into primary cultures derived from mouse skeletal muscle or into myogenic cell lines. We demonstrate that the mouse MLC1F/MLC3F gene has two functional promoters. In primary muscle cultures, a 1200 bp sequence flanking exon 1 (MLC1F) and a 438 bp sequence flanking exon 2 (MLC3F) direct CAT activity in myotubes, but not in myoblasts or in non myogenic 3T6 and CV1 cells. Developmentally regulated expression is also seen with the alpha-cardiac actin (320 bp) and acetylcholine receptor alpha-subunit (850 bp) upstream sequences in the primary culture system. Transfection experiments with myogenic cell lines show different results with a given promoter construct, reflecting possible differences in the levels of regulatory factors between lines. Different muscle gene promoters behave differently in a given cell line, suggesting different regulatory factor requirements between these promoters. Images PMID:2894633

  3. Protein-kinase-Cmu expression correlates with enhanced keratinocyte proliferation in normal and neoplastic mouse epidermis and in cell culture.

    PubMed

    Rennecke, J; Rehberger, P A; Fürstenberger, G; Johannes, F J; Stöhr, M; Marks, F; Richter, K H

    1999-01-05

    In order to gain insight into the biological function of a PKC iso-enzyme, the protein kinase Cmu, we analyzed the expression pattern of this protein in mouse epidermis and keratinocytes in culture. Daily analysis of neonatal mouse epidermis immediately after birth showed a time-dependent reduction in the PKCmu content. Expression of the proliferating-cell nuclear antigen (PCNA), indicative of the proliferative state of cells, was reduced synchronously with PKCmu as the hyperplastic state of the neonatal tissue declined. In epidermal mouse keratinocytes, fractionated according to their maturation state, PKCmu expression was restricted to PCNA-positive basal-cell fractions. In primary cultures of those cells, growth arrest and induction of terminal differentiation by Ca2+ resulted in strongly reduced PKCmu expression, concomitantly with the loss of PCNA expression. Treatment of PMK-R1 keratinocytes with 100 nM of the mitogen 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in activation of PKCmu, reflected by translocation from the cytosolic to the particulate fraction and by shifts in electrophoretic mobility. DNA synthesis was significantly inhibited by the PKCmu inhibitor Goedecke 6976, while Goedecke 6983 did not inhibit PKCmu. Carcinomas generated according to the 2-stage carcinogenesis protocol in mouse skin consistently exhibited high levels of PKCmu. These data correlate PKCmu expression with the proliferative state of murine keratinocytes and point to a role of PKCmu in growth stimulation. A correlation between PKCmu expression and enhanced cell proliferation was also observed for NIH3T3 fibroblasts transfected with and overexpressing human PKCmu.

  4. Na+ dependent glutamate transporters (EAAT1, EAAT2, and EAAT3) in primary astrocyte cultures: effect of oxidative stress.

    PubMed

    Miralles, V J; Martínez-López, I; Zaragozá, R; Borrás, E; García, C; Pallardó, F V; Viña, J R

    2001-12-13

    The Na+ -dependent L-glutamate transporters EAAT1(GLAST), EAAT2 (GLT-1) and EAAT3 (EAAC1) are expressed in primary astrocyte cultures, showing that the EAAT3 transporter is not neuron-specific. The presence of these three transporters was evaluated by RT-PCR, immunoblotting, immunocytochemical techniques, and transport activity. When primary astrocyte cultures were incubated with L-buthionine-(S,R)-sulfoximine (BSO), a selective inhibitor of gamma-glutamylcysteine synthetase, the GSH concentration was significantly lower than in control cultures, but the expression and amount of protein of EAAT1, EAAT2 and EAAT3 and transport of L-glutamate was unchanged. Oxidative stress was created by adding H(2)O(2) or tert.-butyl hydroperoxide (t-bOOH) to the primary astrocyte cultures and cell damage was evaluated by measuring activity of lactate dehydrogenase. Under oxidative stress, GSH levels were significantly lower than in control astrocytes; but the expression and the amount of protein of the three transporters remained unchanged. However, L-glutamate uptake was significantly lower in astrocytes under oxidative conditions when compared to controls. L-Glutamate uptake was not changed in the presence of ascorbate, but was partially recovered in the presence of DTT and GSH ethyl ester. This report emphasizes that oxidative stress and not GSH depletion alters transporter activity without changing transporter expression.

  5. The effects of BmNPV on biochemical changes in primary cultures of Bombyx mori embryonic tissue.

    PubMed

    Matindoost, Leila; Sendi, Jalal Jalali; Soleimanjahi, Hoorieh; Etebari, Kayvan; Rahbarizade, Fateme

    2008-01-01

    The effect of Bombyx mori nuclear polyhedrosis virus (BmNPV) on biochemical changes of TC-100 medium containing 10% fetal bovine serum (FBS) in embryonic primary cultures of silkworm was investigated. The primary cultures that reached 60% confluence were infected by 0.5, 1, and 2-ml viral inoculums (diluted with TC-100 medium representing multiplicity of infection (MOI) of 0.25, 0.5, and 1). Glucose, uric acid, urea, total protein, cholesterol, and alkaline phosphatase were measured in the medium of BmNPV-infected primary cultures. All biochemical compounds showed significant changes. Glucose decreased considerably by about 55 mg/ml, while different concentrations of the virus inoculums did not demonstrate significant differences among them. Total protein had only increased in 2 ml concentration and there were no changes in other concentrations. Uric acid as a by-product accumulated dramatically in all concentrations, while the amount of urea reduced in all treatments and this reduction was more evident in lower concentrations. Cholesterol consumption was high in cultures postinfection, while alkaline phosphatase (ALP) activity decreased in infected cells.

  6. African American Identity and a Theory for Primary Cultural Instructional Design

    ERIC Educational Resources Information Center

    Thomas, Michael K.; Columbus, Marco A.

    2010-01-01

    This article is on the strange confluence of culture, identity, learning, and systemic design. We argue that the work of instructional design is, essentially, work on culture and identity. A person's culture and identity fully and inextricably situate their thought, action, and interaction. For this reason, this inherent situatedness of culture…

  7. Organizational Culture in a Successful Primary School: An Ethnographic Case Study

    ERIC Educational Resources Information Center

    Negis-Isik, Ayse; Gursel, Musa

    2013-01-01

    Even though they are perceived similar from outside, all schools have distinct characteristics and a culture that differ them from other schools. School culture, is one of the important factors that play role in school efficiency and success. The purpose of this study was to examine the culture of a successful school profoundly. This study was a…

  8. Building and Leading a Learning Culture among Teachers: A Case Study of a Shanghai Primary School

    ERIC Educational Resources Information Center

    Haiyan, Qian; Walker, Allan; Xiaowei, Yang

    2017-01-01

    A positive teacher learning culture is important to effect meaningful changes in schools. Literature has established that successful school leaders can build and nurture learning cultures among teachers. However, less is known about how school leaders can shape the culture and make learning conditions happen at the schools in the Chinese education…

  9. Molecular and immunocytochemical characterization of primary neuronal cultures from adult rat brain: Differential expression of neuronal and glial protein markers.

    PubMed

    Ray, Balmiki; Bailey, Jason A; Sarkar, Sumit; Lahiri, Debomoy K

    2009-11-15

    Neurobiological studies using primary neuronal cultures commonly employ fetal-derived neurons, but much less often adult brain-derived neurons. Our goal is to perform morphological and molecular characterization of primary neuronal cultures from adult rat brain, including the relative expression of neuronal and glial cell markers at different time points. We tested the hypothesis that long-term neuronal viability is compatible with glial proliferation in adult neuron culture. We examined neuron culture from adult rat brain, which was maintained at steady state up to 24 days, and characterized them on the basis of cellular, molecular and biochemical properties at different time points of the culture. We identified neuronal and glial cells by both immunocytochemical and western immunoblotting techniques using NSE and Tau as neuronal markers and GFAP as glial protein marker, which revealed the presence of predominantly neuronal cells in the initial phase of the culture and a rise in glial cells from day 12 onwards. Notably, neuronal cells were preserved in the culture along with the glial cells even at day 24. Transfection of the cultured cells with a GFP expression vector and plasmids containing a luciferase reporter gene under the control of two different gene promoters demonstrated DNA transfectability. Taken together, these results suggest a differential expression of neuronal and glial cells at different time points and long-term neuronal viability in the presence of glial proliferation. Such adult neurons serve as a suitable system for the application of neurodegeneration models and for drug target discovery in various brain disorders including Alzheimer's disease.

  10. Fast isolation and expansion of multipotent cells from adipose tissue based on chitosan-selected primary culture.

    PubMed

    Huang, Guo-Shiang; Tseng, Ting-Chen; Dai, Niann-Tzyy; Fu, Keng-Yen; Dai, Lien-Guo; Hsu, Shan-Hui

    2015-10-01

    Adipose-derived adult stem cells (ASCs) have gained much attention because of their multipotency and easy access. Here we describe a novel chitosan-based selection (CS) system instead of the conventional plastic adherence (PA) to obtain the primary ASCs. The minimal amount of adipose tissue for consistent isolation of ASCs is reduced from 10 mL to 5 mL. The selection is based on the specific interaction between cells and chitosan materials, which separate ASCs by forming spheroids during primary culture. The primary culture period was reduced from 4 days to one day and more ASCs (ten-fold expansion) were achieved in a week. The average duration for obtaining 1 × 10(7) cells takes about seven days from 5 mL of adipose tissue, compared to 14 days using the conventional PA method from 10 mL of adipose tissue. The replicative senescence of CS-ASCs is not evident until the fifteenth passage (vs. eighth for the PA-ASCs). The obtained ASCs (CS-ASCs) have less doubling time for the same passage of cells and show greater stemness than those obtained from the conventional PA method (PA-ASCs). Moreover, CS-ASCs undergo trilineage differentiation more effectively than PA-ASCs. The greater differentiation potential of CS-ASCs may be associated with the enrichment and maintenance of CD271 positive cells by chitosan selection of primary culture.

  11. Highly purified hexachlorobenzene induces cytochrome P4501A in primary cultures of chicken embryo hepatocytes

    SciTech Connect

    Mundy, Lukas J.; Jones, Stephanie P.; Crump, Doug; Herve, Jessica C.; Konstantinov, Alex; Utley, Fiona; Potter, David; Kennedy, Sean W.

    2010-11-01

    Some uncertainty exists regarding the purity of hexachlorobenzene (HCB) used in past toxicity studies. It has been suggested that reported toxic and biochemical effects initially attributed to HCB exposure may have actually been elicited by contamination of HCB by polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs). Herein, primary cultures of chicken embryo hepatocytes (CEH) were used to compare the potencies of two lots of reagent-grade hexachlorobenzene (HCB-old [HCB-O] and HCB-new [HCB-N]), highly purified HCB (HCB-P) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) as inducers of ethoxyresorufin O-deethylase (EROD) activity, cytochrome P4501A4 (CYP1A4) messenger ribonucleic acid (mRNA) and CYP1A5 mRNA. The study also compared the EROD- and CYP1A4/5 mRNA-inducing potencies of HCB to the potencies of two mono-ortho substituted polychlorinated biphenyls (PCBs), 2,3,3',4,4'-pentachlorobiphenyl (PCB 105) and 2,3'4,4',5-pentachlorobiphenyl (PCB 118). HCB-O, HCB-N and HCB-P all induced EROD activity and up-regulated CYP1A4 and CYP1A5 mRNAs. Induction was not caused by contamination of HCB with PCDDs or PCDFs. Based upon a comparison of the EC{sub 50} and EC{sub threshold} values for EROD and CYP1A4/5 mRNA concentration-response curves, the potency of HCB relative to the potency of TCDD was 0.0001, and was similar to that of PCB 105 and PCB 118. The maximal EROD activity and CYP1A4/5 mRNA expression differed greatly between HCB and TCDD, and may contribute to an overestimation of the ReP value calculated for highly purified HCB.

  12. Aroclor 1254 increases the genotoxicity of several carcinogens to liver primary cell cultures

    SciTech Connect

    Mendoza-Figueroa, T.; Lopez-Revilla, R.; Villa-Trevino, S.

    1985-01-01

    The genotoxicity of both direct-acting and precarcinogenic chemicals was evaluated in liver primary cell cultures (LPCC) from untreated and Aroclor 1254 (Ar) pretreated rats. Hepatocytes were isolated from partially hepatectomized rats and their DNA was labeled in vitro with (/sup 3/H) dThd; the molecular weight of single-stranded DNA was determined by alkaline sucrose sedimentation. Two parameters of DNA damage were defined: 1) the mean effective dose (ED50), i.e., the carcinogen concentration that decreased the DNA molecular weight to half the original, and 2) the DNA breaking potency (DBP), i.e., the number of breaks per DNA molecule produced by 2 h exposure to 1mM concentration of the chemical. Two hours exposure of LPCC from untreated rats to the direct-acting alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) (6.8-340..mu..M) and to the precarcinogens benzo(a)pyrene (BaP) (0.05-0.33 mM) and dimethylnitrosamine (DMN) (0.45-16 mM) produced a concentration-dependent decrease in the molecular weight of DNA. Pretreatment of rats with Ar decreased significantly the sedimentation velocity of DNA and increased five, three, and two times the DBP of MNNG, BaP, and DMN, respectively. These results show that Ar-pretreatment of rats increases the genotoxicity of both direct-acting and precarcinogenic chemicals and suggest that Ar might increase the genotoxicity of chemical carcinogens perhaps by enhancing their metabolic activation, by producing direct genotoxic effects, or both. Our results also emphasize the carcinogenic risk that the environmental pollution by polychlorinated biphenyls might represent to humans.

  13. Phosphoinositides metabolism in primary culture of dog thyroid cells: Effects of thyrotropin and carbachol

    SciTech Connect

    Taguchi, M.; Field, J.B. )

    1990-04-01

    Thyrotropin (TSH) and carbachol stimulated in a dose-dependent manner the accumulation of 3H-glycerophosphoinositol (GPI), 3H-inositol monophosphate (IP1), 3H-inositol bisphosphate (IP2) and 3H-inositol trisphosphate (IP3) in primary cultures of dog thyroid cells prelabeled with myo-(2-3H)inositol. TSH, 250 mU/mL, stimulated 3H-IP3 level after a 10-minute incubation while 10 mU/mL TSH increased it during a 60-minute incubation. The effect of carbachol was more rapid and greater than that of TSH. Carbachol, 100 mumol/L, elevated 3H-IP3 after a 2-minute incubation and 3H-IP3 formation was increased by as little as 1 mumol/L carbachol. TSH stimulation was observed only if the cells were deprived of TSH for 5 days before being labeled with 3H-inositol. Prolongation of the labeling period or addition of TSH, (Bu)2cAMP or carbachol during the labeling increased 3H-inositol incorporation into polyphoinositides (PIPs). When the cells were labeled without any other addition, control and TSH-stimulated 3H-IP3 levels increased in parallel with 3H-PIP levels. However, TSH or carbachol-stimulated 3H-IP3 levels did not increase in proportion to 3H-PIPs level when the cells were labeled with TSH or (Bu)2cAMP. Thus, the ratio of 3H-IP3/3H-PIPs (both control and TSH or carbachol-stimulated) decreased in the cells labeled with TSH or (Bu)2cAMP, which might reflect TSH stimulation of 3H-inositol incorporation into PIPs pool(s) that do not participate in hormone-induced hydrolysis of PIPs.

  14. Kinetics and autoradiography of high affinity uptake of serotonin by primary astrocyte cultures

    SciTech Connect

    Katz, D.M.; Kimelberg, H.K.

    1985-07-01

    Primary astrocyte cultures prepared from the cerebral cortices of neonatal rats showed significant accumulation of serotonin (5-hydroxytryptamine; (/sup 3/H)-5-HT). At concentrations in the range of 0.01 to 0.7 microM (/sup 3/H)-5-HT, this uptake was 50 to 85% Na+ dependent and gave a Km of 0.40 +/- 0.11 microM (/sup 3/H)-5-HT and a Vmax of 6.42 +/- 0.85 (+/- SEM) pmol of (/sup 3/H)-5-HT/mg of protein/4 min for the Na+-dependent component. In the absence of Na+ the uptake was nonsaturable. Omission of the monoamine oxidase inhibitor pargyline markedly reduced the Na+-dependent component of (/sup 3/H)-5-HT uptake but had a negligible effect on the Na+-independent component. This suggest significant oxidative deamination of serotonin after it has been taken up by the high affinity system, followed by release of its metabolite. The authors estimated that this system enabled the cells to concentrate (/sup 3/H)-5-HT up to 44-fold at an external (/sup 3/H)-5-HT concentration of 10(-7) M. Inhibition of (/sup 3/H)-5-HT uptake by a number of clinically effective antidepressants was also consistent with a specific high affinity uptake mechanism for 5-HT, the order of effectiveness of inhibition being chlorimipramine greater than fluoxetine greater than imipramine = amitriptyline greater than desmethylimipramine greater than iprindole greater than mianserin. Uptake of (/sup 3/H)-5-HT was dependent on the presence of Cl- as well as Na+ in the medium, and the effect of omission of both ions was nonadditive. Varying the concentration of K+ in the media from 1 to 50 mM had a limited effect on (/sup 3/H)-5-HT uptake.

  15. The Antidiabetic Drug Metformin Stimulates Glycolytic Lactate Production in Cultured Primary Rat Astrocytes.

    PubMed

    Westhaus, Adrian; Blumrich, Eva Maria; Dringen, Ralf

    2017-01-01

    Metformin is the most frequently used drug for the treatment of type 2 diabetes in humans. However, only little is known about effects of metformin on brain metabolism. To investigate potential metabolic consequences of an exposure of brain cells to metformin, we incubated rat astrocyte-rich primary cultures with this compound. Metformin in concentrations of up to 30 mM did not acutely compromise the viability of astrocytes, but caused a time- and concentration-dependent increase in cellular glucose consumption and lactate production. For acute incubations in the hour range, the presence of 10 mM metformin doubled the glycolytic flux, while already 1 mM metformin doubled glycolytic flux during incubation for 24 h. In addition to metformin, also other guanidino compounds increased astrocytic lactate production. After 4 h of incubation, half-maximal stimulation of glycolysis was observed for metformin, guanidine and phenformin at concentrations of around 3 mM, 3 mM and 30 µM, respectively. The acute stimulation of glycolytic lactate production by metformin was persistent after removal of extracellular metformin and was also observed, if glucose was absent from the incubation medium or replaced by other hexoses. The metformin-induced stimulation of glycolytic flux was not prevented by compound C, an inhibitor of AMP-dependent protein kinase, nor was it additive to the stimulation of glycolytic flux caused by respiratory chain inhibitors. These data demonstrate that the antidiabetic drug metformin has the potential to strongly activate glycolytic lactate production in brain astrocytes.

  16. Expression profiling of interindividual variability following xenobiotic exposures in primary human hepatocyte cultures

    SciTech Connect

    Goyak, Katy M.O.; Johnson, Mary C.; Strom, Stephen C.; Omiecinski, Curtis J.

    2008-09-01

    To examine the magnitude of human variability across the entire transcriptome after chemical challenge, we profiled gene expression responses to three different prototypic chemical inducers in primary human hepatocyte cultures from ten independent donors. Correlation between basal expression in any two hepatocyte donors ranged from r{sup 2} values of 0.967 to 0.857, and chemical treatment tended to negatively impact correlation between donors. Including anticipated target genes, 10,812, 8373, and 7847 genes were changed in at least one donor by Aroclor 1254 (A1254), di(2-ethylhexyl) phthalate (DEHP), and phenobarbital (PB), respectively. A subset of these gene targets (n = 41) were altered with a high level of reproducibility in at least 9 donors, gene responses that correlated well with literature-reported mechanism of action. Filtering responses to the level of gene subsets clarified the biological impact associated with the respective chemical effectors, in lieu of substantial interindividual variation among donor responses. In these respects, the use of hierarchical clustering methods successfully grouped seven of the ten donors into chemical-specific rather than donor-specific clusters. However, at the whole-genome level, the magnitude of conserved gene expression changes among donors was surprisingly small, with fewer than 50% of the gene responses altered by a single chemical conserved in more than one donor. The use of higher level descriptors, such as those defined by the PANTHER classification system, may enable more consistent categorization of gene expression changes across individuals, as increased reproducibility was identified using this method.

  17. Comparison of calcium ionophore and receptor-activated inositol phosphate formation in primary glial cell cultures.

    PubMed

    Wigginton, S A; Minneman, K P

    1991-11-13

    The possible role of Ca2+ influx in alpha 1-adrenoceptor-stimulated [3H]inositol phosphate [( 3H]InsP) formation was examined in primary cultures of glial cells from 1-day-old rat brain. The Ca2+ ionophore A23187 caused a concentration- and time-dependent increase in [3H]InsP formation similar in magnitude to that caused by norepinephrine (NE). Responses to A23187 and NE were both completely dependent on extracellular Ca2+, with a similar concentration dependence. However, cadmium was more potent in blocking the response to A23187 than to NE. Lanthanum (1 mM) blocked the response to NE, although cobalt (5 mM) did not. The [3H]InsP response to A23187 was not additive with the response to NE or to the muscarinic agonist carbachol, although responses to NE and carbachol were addictive Both A23187 and ionomycin inhibited the additive stimulation caused by a combination of NE and carbachol, and this inhibition was potentiated by cadmium. Ionomycin stimulated [3H]InsP formation at concentrations lower than those inhibiting receptor-mediated responses, and this stimulation was not additive with responses to NE or carbachol. High-performance liquid chromatography separation showed similar patterns of [3H]InsPs formed in response to both Ca2+ ionophore and receptor agonists. These results raise the possibility that receptor-activated Ca2+ influx may be involved in stimulation of [3H]InsP formation in these cells.

  18. Steroidogenesis in primary cultures of neonatal porcine Leydig cells from Duroc and Norwegian Landrace breeds.

    PubMed

    Lervik, S; von Krogh, K; Karlsson, C; Olsaker, I; Andresen, Ø; Dahl, E; Verhaegen, S; Ropstad, E

    2011-10-01

    Breed differences in steroidogenic activity between primary Leydig cells derived from neonatal purebred Duroc and Norwegian Landrace boars were investigated in vitro. Concentrations of testosterone, estradiol, androstenone, cortisol and progesterone produced into the medium were determined. To explore underlying mechanisms the cellular expression of a suite of genes relevant in steroidogenesis was measured using reverse transcription and quantitative PCR (RT-qPCR). Basal steroid concentrations indicated a larger production capacity for steroids in unstimulated Duroc cells. Stimulation of the cells with LH increased steroid hormone secretion significantly in both breeds in a dose dependent manner. Testosterone and androstenone concentrations increased approximately 50- and 15-fold, respectively, whereas concentrations of estradiol, cortisol and progesterone increased to a lesser extent. At levels of maximal LH stimulation, absolute steroid concentrations were higher in Duroc. However, the relative increase in hormone concentrations was significantly lower in Duroc cells for estradiol, progesterone and cortisol when compared to basal levels. LH exposure was associated with a general up-regulation of mRNA levels for steroidogenic genes, stronger in Duroc than in Norwegian Landrace. This was in agreement with the higher absolute concentrations of steroid hormones measured in culture medium from the LH-stimulated Duroc Leydig cells, but did not concur with the fact that the relative increase in hormone production was lower in Duroc than in Norwegian Landrace Leydig cells for some hormones. It was concluded that breed differences in steroid hormone concentrations and gene expression between Norwegian Landrace and Duroc are complex and cannot be explained by a simple mechanism of action.

  19. Neurogenic and Neurotrophic Effects of BDNF Peptides in Mouse Hippocampal Primary Neuronal Cell Cultures

    PubMed Central

    Cardenas-Aguayo, Maria del Carmen; Kazim, Syed Faraz; Grundke-Iqbal, Inge; Iqbal, Khalid

    2013-01-01

    The level of brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, is down regulated in Alzheimer’s disease (AD), Parkinson’s disease (PD), depression, stress, and anxiety; conversely the level of this neurotrophin is increased in autism spectrum disorders. Thus, modulating the level of BDNF can be a potential therapeutic approach for nervous system pathologies. In the present study, we designed five different tetra peptides (peptides B-1 to B-5) corresponding to different active regions of BDNF. These tetra peptides were found to be non-toxic, and they induced the expression of neuronal markers in mouse embryonic day 18 (E18) primary hippocampal neuronal cultures. Additionally, peptide B-5 induced the expression of BDNF and its receptor, TrkB, suggesting a positive feedback mechanism. The BDNF peptides induced only a moderate activation (phosphorylation at Tyr 706) of the TrkB receptor, which could be blocked by the Trk’s inhibitor, K252a. Peptide B-3, when combined with BDNF, potentiated the survival effect of this neurotrophin on H2O2-treated E18 hippocampal cells. Peptides B-3 and B-5 were found to work as partial agonists and as partial antagonists competing with BDNF to activate the TrkB receptor in a dose-dependent manner. Taken together, these results suggest that the described BDNF tetra peptides are neurotrophic, can modulate BDNF signaling in a partial agonist/antagonist way, and offer a novel therapeutic approach to neural pathologies where BDNF levels are dysregulated. PMID:23320097

  20. Protective effect of dieckol against chemical hypoxia-induced cytotoxicity in primary cultured mouse hepatocytes.

    PubMed

    Jeon, Yu Jin; Kim, Hyoung Seok; Song, Kyung-Sik; Han, Ho Jae; Park, Soo Hyun; Chang, Woochul; Lee, Min Young

    2015-04-01

    Hepatic ischemic injury is a major complication arising from liver surgery, transplantation, or other ischemic diseases, and both reactive oxygen species (ROS) and pro-inflammatory mediators play the role of key mediators in hepatic ischemic injury. In this study, we examined the effect of dieckol in chemical hypoxia-induced injury in mouse hepatocytes. Cell viability was significantly decreased after treatment with cobalt chloride (CoCl2), a well-known hypoxia mimetic agent in a time- and dose-dependent manner. Pretreatment with dieckol before exposure to CoCl2 significantly attenuated the CoCl2-induced decrease of cell viability. Additionally, pretreatment with dieckol potentiated the CoCl2-induced decrease of Bcl-2 expression and attenuated the CoCl2-induced increase in the expression of Bax and caspase-3. Treatment with CoCl2 resulted in an increased intracellular ROS generation, which is inhibited by dieckol or N-acetyl cysteine (NAC, a ROS scavenger), and p38 MAPK phosphorylation, which is also blocked by dieckol or NAC. In addition, dieckol and SB203580 (p38 MAPK inhibitor) increased the CoCl2-induced decrease of Bcl-2 expression and decreased the CoCl2-induced increase of Bax and caspase-3 expressions. CoCl2-induced decrease of cell viability was attenuated by pretreatment with dieckol, NAC, and SB203580. Furthermore, dieckol attenuated CoCl2-induced COX-2 expression. Similar to the effect of dieckol, NAC also blocked CoCl2-induced COX-2 expression. Additionally, CoCl2-induced decrease of cell viability was attenuated not only by dieckol and NAC but also by NS-398 (a selective COX-2 inhibitor). In conclusion, dieckol protects primary cultured mouse hepatocytes against CoCl2-induced cell injury through inhibition of ROS-activated p38 MAPK and COX-2 pathway.

  1. Primary airway epithelial cell culture and asthma in children-lessons learnt and yet to come.

    PubMed

    McLellan, Kirsty; Shields, Mike; Power, Ultan; Turner, Steve

    2015-12-01

    Until recently the airway epithelial cell (AEC) was considered a simple barrier that prevented entry of inhaled matter into the lung parenchyma. The AEC is now recognized as having an important role in the inflammatory response of the respiratory system to inhaled exposures, and abnormalities of these responses are thought to be important to asthma pathogenesis. This review first explores how the challenges of studying nasal and bronchial AECs in children have been addressed and then summarizes the results of studies of primary AEC function in children with and without asthma. There is good evidence that nasal AECs may be a suitable surrogate for the study of certain aspects of bronchial AEC function, although bronchial AECs remain the gold standard for asthma research. There are consistent differences between children with and without asthma for nasal and bronchial AEC mediator release following exposure to a range of pro-inflammatory stimulants including interleukins (IL)-1β, IL-4, and IL-13. However, there are inconsistencies between studies, e.g., release of IL-6, an important pro-inflammatory cytokine, is not increased in children with asthma relative to controls in all studies. Future work should expand current understanding of the "upstream" signalling pathways in AEC, study AEC from children before the onset of asthma symptoms and in vitro models should be developed that replicate the in vivo status more completely, e.g., co-culture with dendritic cells. AECs are difficult to obtain from children and collaboration between centers is expected to yield meaningful advances in asthma understanding and ultimately help deliver novel therapies.

  2. v-src transformation of rat embryo fibroblasts. Inefficient conversion to anchorage-independent growth involves heterogeneity of primary cultures

    PubMed Central

    1994-01-01

    To clarify whether a single oncogene can transform primary cells in culture, we compared the transforming effect of a recombinant retrovirus (ZSV) containing the v-src gene in rat embryo fibroblasts (REFs) to that in the rat cell line 3Y1. In the focus assay, REFs exhibited resistance to transformation as only six foci were observed in the primary cultures as opposed to 98 in 3Y1 cells. After G418 selection, efficiency of transformation was again somewhat lower with REFs compared to that with 3Y1 cells, but the number of G418-resistant REF colonies was much greater than the number of foci in REF cultures. Furthermore, while 98% of G418-resistant colonies of ZSV-infected REFs were morphologically transformed, only 25% were converted to anchorage- independent growth, as opposed to 100% conversion seen in ZSV-infected 3Y1 cells. The poor susceptibility of REFs to anchorage-independent transformation did not involve differences in expression and subcellular distribution of p60v-src, or its kinase activity in vitro and in vivo. It rather reflected a property of the primary cultures, as cloning of REFs before ZSV infection demonstrated that only 2 out of 6 REF clones tested were permissive for anchorage-independent growth. The nonpermissive phenotype was dominant over the permissive one in somatic hybrid cells, and associated with organized actin filament bundles and a lower growth rate, both before and after ZSV infection. These results indicate that the poor susceptibility of REFs to anchorage-independent transformation by p60v-src reflects the heterogeneity of the primary cultures. REFs can be morphologically transformed by p60v-src with high efficiency but only a small fraction is convertible to anchorage- independent growth. REF resistance seems to involve the presence of a suppressor factor which may emerge from REF differentiation during embryonic development. PMID:8034746

  3. Benzo(a)pyrene metabolism in primary cultures of mouse epidermal cells and untransformed and transformed epidermal cell lines.

    PubMed

    DiGiovanni, J; Miller, D R; Singer, J M; Viaje, A; Slaga, T J

    1982-07-01

    The metabolism of [3H]benzo(a)pyrene [B(a)P] by cultures of primary mouse epidermal cells and untransformed and transformed epidermal cell lines was investigated. All three cell types effectively metabolized [3H]B(a)P. The major organic solvent-extractable metabolites found intracellularly in primary cultures were trans-7,8-dihydro-7,8-dihydroxybenzo(a)pyrene and 3-hydroxybenzo(a)pyrene, although quantities of 9-hydroxybenzo(a)pyrene, trans-9,10-dihydro-9,10-dihydroxybenzo(a)pyrene, and quinones also were present. The major organic solvent-soluble metabolites found in the extracellular medium were trans-9,10-dihydro-9,10-dihydroxybenzo(a)pyrene and trans-7,8-dihydro-7,8-dihydroxybenzo(a)pyrene, with smaller quantities of unconjugated phenols and quinones. The major water-soluble metabolites found in the extracellular medium were conjugated with glucuronic acid [primarily 3-hydroxybenzo(a)pyrene and several quinones]. No sulfate conjugates of [3H]B(a)P metabolites were detected. [3H]B(a)P metabolism was similar in cultures of untransformed and transformed epidermal cell lines but differed from the primary cultures. The major intracellular and extracellular organic solvent-soluble metabolites were diols. Little or no unconjugated phenols were detected. Both the untransformed and transformed epidermal cell lines converted [3H]B(a)P to water-soluble metabolites, primarily glucuronide conjugates. In contrast to the primary cells, a major pathway of trans-7,8-dihydro-7,8-dihydroxybenzo(a)pyrene metabolism in the untransformed and transformed cell lines was a glucuronide conjugate. Primary mouse epidermal cells provide an important model system for studying factors affecting the activation and detoxification of hydrocarbon carcinogens.

  4. Collaborative Management of Neurocognitive Disorders in Primary Care: Explorations of an Attempt at Culture Change

    PubMed Central

    Mehl-Madrona, Lewis; Mainguy, Barbara

    2017-01-01

    Introduction: Minor neurocognitive disorder (MiND; previously mild cognitive impairment) is a transitional zone between normal cognitive function and early stages of major neurocognitive disorder (previously called dementia). Of people with MiND, 5% to 10% progress to major neurocognitive disorder. Simple interventions such as memory activities, balance exercises, and anti-inflammatory diets have been shown to improve cognitive ability. Also, education and support in group settings have proved beneficial for patients with MiND. Design: Survey evaluation of outcomes of geriatric consultation and prospective educational study. Main Outcome Measures: We collaborated with an academic training program to introduce into primary care the ideas of educational activities and participation in group medical care for people with MiND. Educational programs were developed and presented to family medicine residents and practicing physicians, and their knowledge was assessed before and after education. Results: Two group programs were implemented: one at our hospital and one at a local skilled nursing facility. These were initially envisioned as time-limited, but participants insisted on their continuance. Thirty-two different patients attended the groups for at least six sessions. Participants enthusiastically reported positive change on qualitative interviews and showed improvement in cognition, balance, and self-esteem. Family medicine residents and practicing physicians both shifted toward lifestyle medicine and significantly changed their views on the efficacy of treatments. Despite these activities, community physicians making referrals for geriatric consultations did not change their discussions with patients and families about exercise, diet, cognitive enhancement, and socialization for MiND. Conclusion: Group visits that emphasized support for increased exercise, improved diet, more movement and balance, and cognitive enhancement appear to please and benefit patients with

  5. Representative mammalian cell culture test materials for assessment of primary recovery technologies: a rapid method with industrial applicability.

    PubMed

    Popova, Daria; Stonier, Adam; Pain, David; Titchener-Hooker, Nigel J; Farid, Suzanne S

    2015-01-01

    Mammalian cell culture material is often difficult to produce accurately and reproducibly for downstream studies. This article presents a methodology for the creation of a set of cell culture test materials where key variables including cell density, cell viability, product, and the host cell protein (HCP) load can be manipulated individually. The methodology was developed using a glutamine synthetase Chinese hamster ovary cell line cultured at 5-L and 70-L scales. Cell concentration post-cell growth was manipulated using tangential flow filtration to generate a range of target cell densities of up to 100 × 10(6) cells/mL. A method to prepare an apoptotic cell stock to achieve target viabilities of 40-90% is also described. In addition, a range of IgG1 and HCP concentrations was achieved. The results illustrate that the proposed methodology is able to mimic different cell culture profiles by decoupling the control of the key variables. The cell culture test materials were shown to be representative of typical cell culture feed material in terms of particle size distribution and HCP population. This provides a rapid method to create the required feeds for assessing the feasibility of primary recovery technologies designed to cope with higher cell density cultures.

  6. Primary culture of glial cells from mouse sympathetic cervical ganglion: a valuable tool for studying glial cell biology.

    PubMed

    de Almeida-Leite, Camila Megale; Arantes, Rosa Maria Esteves

    2010-12-15

    Central nervous system glial cells as astrocytes and microglia have been investigated in vitro and many intracellular pathways have been clarified upon various stimuli. Peripheral glial cells, however, are not as deeply investigated in vitro despite its importance role in inflammatory and neurodegenerative diseases. Based on our previous experience of culturing neuronal cells, our objective was to standardize and morphologically characterize a primary culture of mouse superior cervical ganglion glial cells in order to obtain a useful tool to study peripheral glial cell biology. Superior cervical ganglia from neonatal C57BL6 mice were enzymatically and mechanically dissociated and cells were plated on diluted Matrigel coated wells in a final concentration of 10,000cells/well. Five to 8 days post plating, glial cell cultures were fixed for morphological and immunocytochemical characterization. Glial cells showed a flat and irregular shape, two or three long cytoplasm processes, and round, oval or long shaped nuclei, with regular outline. Cell proliferation and mitosis were detected both qualitative and quantitatively. Glial cells were able to maintain their phenotype in our culture model including immunoreactivity against glial cell marker GFAP. This is the first description of immunocytochemical characterization of mouse sympathetic cervical ganglion glial cells in primary culture. This work discusses the uses and limitations of our model as a tool to study many aspects of peripheral glial cell biology.

  7. Cell Culture Made Easy.

    ERIC Educational Resources Information Center

    Dye, Frank J.

    1985-01-01

    Outlines steps to generate cell samples for observation and experimentation. The procedures (which use ordinary laboratory equipment) will establish a short-term primary culture of normal mammalian cells. Information on culture vessels and cell division and a list of questions to generate student interest and involvement in the topics are…

  8. FSH-initiated differentiation of newt spermatogonia to primary spermatocytes in germ-somatic cell reaggregates cultured within a collagen matrix.

    PubMed

    Ito, R; Abé, S I

    1999-03-01

    We previously cultured fragments of newt testes in chemically defined media and showed that mammalian follicle-stimulating hormone (FSH) stimulates proliferation of spermatogonia as well as their differentiation into primary spermatocytes (Ji et al., 1992; Abe and Ji, 1994). Next, we indicated in cultures composed of spermatogonia and somatic cells (mainly Sertoli cells) that FSH stimulates germ cell proliferation via Sertoli cells (Maekawa et al., 1995). However, the spermatogonia did not differentiate into primary spermatocytes, but instead died. In the present study, we embedded large reaggregates of spermatogonia and somatic cells (mainly Sertoli cells) within a collagen matrix and cultured the reaggregates on a filter that floated on chemically defined media containing FSH; in this revised culture system, spermatogonia proliferated and differentiated into primary spermatocytes. The viability and percentage of germ cells differentiating into primary spermatocytes were proportional to the percentage of somatic cells in the culture, indicating that differentiation of spermatogonia into primary spermatocytes is mediated by Sertoli cells.

  9. Proteomic profile of primary isolated rat mesangial cells in high-glucose culture condition and decreased expression of PSMA6 in renal cortex of diabetic rats.

    PubMed

    Li, Zhiguo; Zhang, Haojun; Dong, Xi; Burczynski, Frank J; Choy, Patrick; Yang, Fang; Liu, Hui; Li, Ping; Gong, Yuewen

    2010-08-01

    Diabetic nephropathy (DN) is one of the most important complications of diabetic patients and is characterized histologically by an accumulation of extracellular matrix (ECM) protein in the glomerular mesangium. Therefore, mesangial cells likely play an important role in the development of diabetic nephropathy. Here, we employed proteomic techniques to investigate the protein profile of rat mesangial cells under high-glucose culture conditions. Primary isolated rat glomerular mesangial cells were cultured under different concentrations of glucose (5.4 mmol.L-1 for normal control and 30 mmol.L-1 for high glucose) for 0, 8, 16, and 72 h, as well as for 25 days. Cellular total proteins were isolated from these cells and employed for two-dimensional gel electrophoresis (2-DE). Differentially expressed proteins were identified by matrix-assisted laser desorption - ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and some of these proteins were documented in rat models of diabetes by Western blot. Rat mesangial cells were successfully isolated in the laboratory and their proliferation rates were significantly inhibited by high glucose. Two-dimensional gel electrophoresis analyses revealed 28 differentially expressed protein spots between the normal and high-glucose groups. After MALDI-TOF-MS analysis, all 28 protein spots were successfully identified with the peptide mass fingerprint (PMF) method. Representatively, SOD1, PCBP1 and PSMA6 were validated by Western blot analysis following protein extractions from the normal and high-glucose groups. Abundance of these proteins was consistent with that found in 2-DE. Moreover, expression of SOD1, PCBP1, and PSMA6 in renal cortex was further examined in two rat models of diabetes (streptozotocin-induced and spontaneous OLETF diabetic models). Abundance of SOD1 and PCBP1 proteins did not show any significant difference between normal control and diabetic rats. However, abundance of the PSMA6 protein was significantly

  10. Primary amenorrhea in adolescent girls: normal coitus or not? Always take a look in the physician's office

    PubMed Central

    2014-01-01

    Background Primary care physicians are frequently faced with the challenge of evaluating primary amenorrhea in adolescent girls. Approximately 15% of these women have abnormal genital examination, with Müllerian agenesis being the second most frequent cause. We report two cases of adolescents with Müllerian agenesis that presented to a tertiary adolescent medicine center with primary amenorrhea and the very rare sexual phenomenon of urethral coitus. The aim of this report is to emphasize the importance of performing a genital examination in girls who present with amenorrhea in the primary care setting, even if ‘normal’ vaginal sexual activity is assumed. Case presentations A 19-year-old Caucasian and a 16-year-old Filipino girl presented to a tertiary adolescent medicine center with primary amenorrhea and a history of ‘normal’ vaginal coitus. Investigation revealed Müllerian agenesis in association with urethral coitus in both cases; neither patient suffered significant urethral damage to require urethra reconstruction. However, the first adolescent had recurrent pyelonephritis and renal scarring and the second had dysuria. To the best of our knowledge, Case 1 also represents the second reported case of pituitary prolactinoma in association with Müllerian agenesis. The first adolescent underwent a hernia repair and vaginoplasty, whereas the second had vaginal dilatations. Conclusion Our cases highlight the need for careful assessment of the external genitalia and vagina patency in all girls with amenorrhea, even if they report ‘normal’ vaginal sexual activity. Early identification of anatomic disorders such as Müllerian agenesis, will allow provision of proper care according to the patient’s needs and the existing abnormalities, and prevention of rare, unintentional but potentially physically and emotionally harmful, patterns of sexual intercourse. PMID:24507015

  11. How to change organisational culture: Action research in a South African public sector primary care facility

    PubMed Central

    De Sa, Angela; Christodoulou, Maria

    2016-01-01

    Background Organisational culture is a key factor in both patient and staff experience of the healthcare services. Patient satisfaction, staff engagement and performance are related to this experience. The department of health in the Western Cape espouses a values-based culture characterised by caring, competence, accountability, integrity, responsiveness and respect. However, transformation of the existing culture is required to achieve this vision. Aim To explore how to transform the organisational culture in line with the desired values. Setting Retreat Community Health Centre, Cape Town, South Africa. Methods Participatory action research with the leadership engaged with action and reflection over a period of 18 months. Change in the organisational culture was measured at baseline and after 18 months by means of a cultural values assessment (CVA) survey. The three key leaders at the health centre also completed a 360-degree leadership values assessment (LVA) and had 6 months of coaching. Results Cultural entropy was reduced from 33 to 13% indicating significant transformation of organisational culture. The key driver of this transformation was change in the leadership style and functioning. Retreat health centre shifted from a culture that emphasised hierarchy, authority, command and control to one that established a greater sense of cohesion, shared vision, open communication, appreciation, respect, fairness and accountability. Conclusion Transformation of organisational culture was possible through a participatory process that focused on the leadership style, communication and building relationships by means of CVA and feedback, 360-degree LVA, feedback and coaching and action learning in a co-operative inquiry group. PMID:27608671

  12. Dynamic Interplay of Flow and Collagen Stabilizes Primary Hepatocytes Culture in a Microfluidic Platform

    PubMed Central

    Hegde, Manjunath; Jindal, Rohit; Bhushan, Abhinav; Bale, Shyam Sundhar; McCarty, William J; Golberg, Inna; Usta, O. Berk; Yarmush, Martin L.

    2014-01-01

    The creation of stable flow cultures of hepatocytes is highly desirable for the development of platforms for drug toxicity screening, bio-artificial liver support devices, and models for investigating liver physiology and pathophysiology. Given that hepatocytes cultured using the collagen overlay or ‘sandwich’ configuration maintain a wide range of differentiated functions, we describe a simple method for adapting this culture configuration within a microfluidic device. The device design consists of a porous membrane sandwiched between two layers of PDMS resulting in a two-chambered device. In the bottom chamber, hepatocytes are cultured in the collagen sandwich configuration, while the top chamber is accessible for flow. We demonstrate that hepatocytes cultured under flow exhibit higher albumin and urea secretion, and induce cytochrome P450 1A1 activity in comparison to static cultures. Furthermore, over two weeks, hepatocytes cultured under flow show a well-connected cellular network with bile canaliculi formation, whereas static cultures result in the formation of gaps in the cellular network that progressively increase over time. Although enhanced functional response of hepatocytes cultured under flow has been observed in multiple prior studies, the exact mechanism for this flow induced effect remains unknown. In our work, we identified that hepatocytes secrete higher level of collagen in the flow cultures; inhibiting collagen secretion within the flow cultures reduced albumin secretion and restored the appearance of gaps in the cellular network similar to the static cultures. These results demonstrate the importance of the increased collagen secretion by hepatocytes cultured under flow as a mechanism to maintain well-connected cellular network and differentiated function. PMID:24770663

  13. Dynamic interplay of flow and collagen stabilizes primary hepatocytes culture in a microfluidic platform.

    PubMed

    Hegde, Manjunath; Jindal, Rohit; Bhushan, Abhinav; Bale, Shyam Sundhar; McCarty, William J; Golberg, Inna; Usta, O Berk; Yarmush, Martin L

    2014-06-21

    The creation of stable flow cultures of hepatocytes is highly desirable for the development of platforms for drug toxicity screening, bio-artificial liver support devices, and models for investigating liver physiology and pathophysiology. Given that hepatocytes cultured using the collagen overlay or in 'sandwich' configuration maintain a wide range of differentiated functions, we describe a simple method for adapting this culture configuration within a microfluidic device. The device design consists of a porous membrane sandwiched between two layers of PDMS resulting in a two-chambered device. In the bottom chamber, hepatocytes are cultured in the collagen sandwich configuration, while the top chamber is accessible for flow. We demonstrate that hepatocytes cultured under flow exhibit higher albumin and urea secretions and induce cytochrome P450 1A1 activity in comparison to static cultures. Furthermore, over two weeks, hepatocytes cultured under flow show a well-connected cellular network with bile canaliculi formation, whereas static cultures show formation of gaps in the cellular network that progressively increase over time. Although enhanced functional response of hepatocytes cultured under flow has been observed in multiple prior studies, the exact mechanism for this flow induced effect remains unknown. In our work, we identified that hepatocytes secrete a higher level of collagen in the flow cultures; inhibiting collagen secretion within the flow cultures reduced albumin secretion and restored the appearance of gaps in the cellular network similar to the static cultures. These results demonstrate the importance of the increased collagen secretion by hepatocytes cultured under flow as a mechanism to maintain a well-connected cellular network and a differentiated function.

  14. Anticancer drugs exert differential apoptotic and cytotoxic effects on glioblastoma primary cultures with various EGFR and bcl-2 profiles.

    PubMed

    Pédeboscq, Stéphane; L'Azou, Béatrice; Passagne, Isabelle; De Giorgi, Francesca; Ichas, François; Liguoro, Dominique; Pometan, Jean-Paul; Cambar, Jean

    2009-01-01

    The aim of this study was to determine the apoptotic and cytotoxic effects induced on glioblastoma cells by various anticancer agents that possess different mechanisms of action (alkylating drugs, anti-EGFR (Epidermal Growth Factor receptor), proteasome inhibitor). Primary cell cultures were obtained from patients who underwent surgery for their glioblastoma. The cytotoxic effects of drugs were determined by MTT (dimethylthiazolyl diphenyl tetrazolium bromide) assay and apoptosis was evaluated by measuring mitochondrial potential by flow cytometry. Biological markers (EGFR, bcl-2) were studied by a immunoblotting technique to find out predictive markers of response. We found a large interindividual sensitivity, thus confirming the interest of the primary cultures. New proteasome inhibitor bortezomib had considerable cytotoxic and apoptotic potential in glioblastoma, even at very low concentrations. Moreover, the characterization of patients' cells for EGFR and bcl-2 status could constitute an interest, with the evaluation of other markers, in the study of expected chemotherapy response.

  15. Kit ligand promotes the transition from primordial to primary follicles after in vitro culture of ovine ovarian tissue.

    PubMed

    Cavalcante, A Y P; Gouveia, B B; Barberino, R S; Lins, T L B G; Santos, L P; Gonçalves, R J S; Celestino, J J H; Matos, M H T

    2016-08-01

    This study evaluated the effects of kit ligand (KL) on the morphology and development of ovine preantral follicles (fresh control) and after 7 days of in vitro culture in α-Minimal Essential Medium (α-MEM; control medium) or the presence of KL (1, 10, 50, 100 or 200 ng/ml). There was an increase in the percentage of primary follicles at the concentration of 100 ng/ml KL, compared with the fresh control, control medium (α-MEM) and the other KL concentrations. Follicle diameter was significantly higher than the control medium only at concentrations of 50 and 100 ng/ml KL. In conclusion, 100 ng/ml KL promoted the transition from primordial to primary follicles (follicular activation) after in vitro culture of ovine ovarian tissue.

  16. Effect of aging on GHRF-induced growth hormone release from anterior pituitary cells in primary culture

    SciTech Connect

    Spik, K.W.; Boyd, R.L.; Sonntag, W.E.

    1991-03-01

    Five criteria were developed to validate the primary cell culture model for comparison of GRF-induced release of growth hormone in pituitary tissue from aging animals. Pituitaries from young (5-mo), middle-aged (14-mo), and old (24-mo) male Fischer 344 rats were dispersed using either trypsin/trypsin inhibitor or dispase and compared with respect to the number of pituitary cells recovered, cell viability, 3H-leucine incorporation into total protein, time course for recovery of optimal response to GRF, and the dose-relationship for GRF-induced release of growth hormone 2, 4, and 6 days after dispersal. Results indicated that direct comparison of cellular responses between tissues from young, middle-aged, and old rats in primary cell culture is confounded by variations in time for recovery of optimal responses, the effects of the enzymes used for dispersal, and the methods used to express the data.

  17. (/sup 3/H) spiperone binding sites in rat primary cultures, C6 glioma, and B104 neuroblastoma

    SciTech Connect

    Severson, J.A.; de Vellis, J.S.; Finch, C.E.

    1980-01-01

    Binding sites for (/sup 3/H) spiperone were detected on membranes from primary glial cultures from neonatal rat cortex and striatum and from the C6 glioma cells. (/sup 3/H) spiperone binding was displacable by d-butaclamol. However, competition studies suggest that (/sup 3/H) spiperone binding to primary glial cultures was mainly to serotonergic sites, and binding to the C6 glioma was alpha-adrenergic. No specific binding was detected to membranes from B104 neuroblastoma cells. Although (/sup 3/H) spiperone binding to glial sites in whole striatum under generally used conditions is small (about 10%), the striatal glial hyperactivity which is often associated with neuronal degeneration could lead to an overestimation of presumed neuronal binding sites for dopaminergic ligands.

  18. Effects of unilateral topical administration of 0.5% tropicamide on anterior segment morphology and intraocular pressure in normal cats and cats with primary congenital glaucoma

    PubMed Central

    Gomes, Filipe Espinheira; Bentley, Ellison; Lin, Ting-Li; McLellan, Gillian J.

    2012-01-01

    Objective To determine the effects of topical 0.5% tropicamide on anterior segment morphology (ASM) and intraocular pressure (IOP) in normal and glaucomatous cats. Animals used Normal cats and cats with inherited primary congenital glaucoma (PCG). Procedures Control IOP curves were performed in untreated normal and PCG cats. In the first experiment, tropicamide was applied OD in eight normal and nine PCG cats. IOP and pupillary diameter (PD) were measured at 0, 30, and 60 min, then hourly until 8 h post-treatment. In a second experiment, six normal and seven PCG cats received tropicamide OD. High-resolution ultrasound images were obtained at 0, 1, 5, and 10 h post-treatment to measure ASM changes. IOP and PD were measured OD at 0, 1, 2, 3, 5, 7, and 9 h. Results In untreated normal cats IOP OU decreased throughout the day. In PCG cats IOP OU had wide fluctuations over time. In normal cats IOP response varied in the treated eye but did not change significantly in untreated eyes. IOP significantly increased from baseline in both eyes of all treated PCG cats. Increases in IOP were associated with some ASM changes. Cats with PCG had a significantly smaller angle recess areas, diminished ciliary clefts and decreased iris-lens contact. ASM changes were not strongly correlated with IOP in all cats. Conclusions The ASM of PCG cats is markedly different from normal cats, and clinically significant increases in IOP OU occur in cats with PCG after tropicamide treatment. The mechanism for this increase remains unclear. PMID:21923827

  19. Comparison of the hepatotoxicity of toxin T-514 of Karwinskia humboldtiana and its diastereoisomer in primary liver cell cultures.

    PubMed

    Garza-Ocañas, L; Jiang, T; Acosta, D; Torres-Alanis, O; Waksman de Torres, N; Piñeyro-Lopez, A

    1994-10-01

    Toxin T-514 of Karwinskia humboldtiana has been demonstrated to be hepatotoxic in vivo and in vitro. Recently a diastereoisomer of T-514 has been isolated. In the present study we have evaluated and compared the in vitro hepatoxicity of the diastereoisomer of T-514 using primary cultures of rat hepatocytes. Cytotoxicity was evaluated by release of cytoplasmic enzyme lactate dehydrogenase (LDH), and mitochondrial metabolic function (MTT reduction). The diastereoisomer was shown to be almost as hepatoxic in vitro as toxin T-514.

  20. Gene expression profiling and differentiation assessment in primary human hepatocyte cultures, established hepatoma cell lines, and human liver tissues

    SciTech Connect

    Olsavsky, Katy M.; Page, Jeanine L.; Johnson, Mary C.; Zarbl, Helmut; Strom, Stephen C.; Omiecinski, Curtis J. . E-mail: cjo10@psu.edu

    2007-07-01

    Frequently, primary hepatocytes are used as an in vitro model for the liver in vivo. However, the culture conditions reported vary considerably, with associated variability in performance. In this study, we characterized the differentiation character of primary human hepatocytes cultured using a highly defined, serum-free two-dimensional sandwich system, one that configures hepatocytes with collagen I as the substratum together with a dilute extracellular matrix (Matrigel{sup TM}) overlay combined with a defined serum-free medium containing nanomolar levels of dexamethasone. Gap junctional communication, indicated by immunochemical detection of connexin 32 protein, was markedly enhanced in hepatocytes cultured in the Matrigel sandwich configuration. Whole genome expression profiling enabled direct comparison of liver tissues to hepatocytes and to the hepatoma-derived cell lines, HepG2 and Huh7. PANTHER database analyses were used to identify biological processes that were comparatively over-represented among probe sets expressed in the in vitro systems. The robustness of the primary hepatocyte cultures was reflected by the extent of unchanged expression character when compared directly to liver, with more than 77% of the probe sets unchanged in each of the over-represented categories, representing such genes as C/EBP{alpha}, HNF4{alpha}, CYP2D6, and ABCB1. In contrast, HepG2 and Huh7 cells were unchanged from the liver tissues for fewer than 48% and 55% of these probe sets, respectively. Further, hierarchical clustering of the hepatocytes, but not the cell lines, shifted from donor-specific to treatment-specific when the probe sets were filtered to focus on phenobarbital-inducible genes, indicative of the highly differentiated nature of the hepatocytes when cultured in a highly defined two-dimensional sandwich system.

  1. Cryopreservation and in vitro culture of primary cell types from lung tissue of a stranded pygmy sperm whale (Kogia breviceps).

    PubMed

    Annalaura Mancia; Spyropoulos, Demetri D; McFee, Wayne E; Newton, Danforth A; Baatz, John E

    2012-01-01

    Current models for in vitro studies of tissue function and physiology, including responses to hypoxia or environmental toxins, are limited and rely heavily on standard 2-dimensional (2-D) cultures with immortalized murine or human cell lines. To develop a new more powerful model system, we have pursued methods to establish and expand cultures of primary lung cell types and reconstituted tissues from marine mammals. What little is known about the physiology of the deep-sea diving pygmy sperm whale (PSW), Kogia breviceps, comes primarily from stranding events that occur along the coast of the southeastern United States. Thus, development of a method for preserving live tissues and retrieving live cells from deceased stranded individuals was initiated. This report documents successful cryopreservation of PSW lung tissue. We established in vitro cultures of primary lung cell types from tissue fragments that had been cryopreserved several months earlier at the stranding event. Dissociation of cryopreserved lung tissues readily provides a variety of primary cell types that, to varying degrees, can be expanded and further studied/manipulated in cell culture. In addition, PSW-specific molecular markers have been developed that permitted the monitoring of fibroblast, alveolar type II, and vascular endothelial cell types. Reconstitution of 3-D cultures of lung tissues with these cell types is now underway. This novel system may facilitate the development of rare or disease-specific lung tissue models (e.g., to test causes of PSW stranding events and lead to improved treatments for pulmonary hypertension or reperfusion injury in humans). Also, the establishment of a "living" tissue bank biorepository for rare/endangered species could serve multiple purposes as surrogates for freshly isolated samples.

  2. Differential stimulation of neurotrophin release by the biocompatible nano-material (carbon nanotube) in primary cultured neurons.

    PubMed

    Kim, Yun Gi; Kim, Jong Wan; Pyeon, Hee Jang; Hyun, Jung Keun; Hwang, Ji-Young; Choi, Seong-Jun; Lee, Ja-Yeon; Deák, Ferenc; Kim, Hae-Won; Lee, Young Il

    2014-01-01

    In order to develop novel, effective therapies for central nervous system regeneration, it is essential to better understand the role of neurotrophic factors and to design, accordingly, better artificial scaffolds to support both neurite outgrowth and synapse formation. Both nerve growth factor and brain-derived neurotrophic factor are major factors in neural survival, development, synaptogenesis, and synaptic connectivity of primary cultured neurons. As a prime candidate coating material for such neural cultures, carbon nanotubes offer unique structural, mechanical, and electrical properties. In this study, carbon nanotubes coated glass-coverslips were used as the matrix of a primary neural culture system used to investigate the effects of carbon nanotubes on neurite outgrowth and nerve growth factor/brain-derived neurotrophic factor release and expression. For these purposes, we performed comparative analyses of primary cultured neurons on carbon nanotubes coated, non-coated, and Matrigel-coated coverslips. The morphological findings showed definite carbon nanotubes effects on the neurite outgrowths and synaptogenic figures in both cortical and hippocampal neurons when compared with the non-coated negative control. Although the carbon nanotubes did not change neurotrophin expression levels, it stimulated brain-derived neurotrophic factor release into the media from both types of neurons. Accordingly, we suggest a different mechanism of action between carbon nanotubes and Matrigel in relation to the specific neurotrophic factors. Since carbon nanotubes supply long-term extracellular molecular cues for the survival and neurite outgrowths of cultured neurons, the results from this study will contribute to an understanding of carbon nanotubes biological effects and provide new insight into their role in the secretion of neurotrophic factors.

  3. The clash of medical civilizations: experiencing "primary care" in a neoliberal culture.

    PubMed

    McKenna, Brian

    2012-12-01

    An anthropologist describes how he found himself at the vortex of a "clash of medical civilizations:" neoliberalism and the international primary health care movement. His involvement in a $6 million social change initiative in medical education became a basis to unlock the hidden tensions, contradictions and movements within the "primary care" phenomenon. The essay is structured on five ethnographic stories, situated on a continuum from "natural" species-level primary care to "unnatural" neoliberal primary care. Food is an element of all tales. Taking the long view of history/prehistory permits us to better recognize ideological distortions in order to more capably transform medicine.

  4. Activation of antioxidant response element in mouse primary cortical cultures with sesquiterpene lactones isolated from Tanacetum parthenium

    PubMed Central

    Fischedick, Justin T; Standiford, Miranda; Johnson, Delinda A.; De Vos, Ric C.H.; Todorović, Slađana; Banjanac, Tijana; Verpoorte, Rob; Johnson, Jeffrey A.

    2012-01-01

    Tanacetum parthenium (Asteraceae) produces biologically active sesquiterpene lactones (SL). Nuclear factor E2-related factor 2 (Nrf2) is a transcription factor known to activate a series of genes termed the antioxidant response element (ARE). Activation of the Nrf2/ARE may be useful for the treatment of neurodegenerative disease. In this study we isolated 11 sesquiterpene lactones from T. parthenium with centrifugal partition chromatography and semi-preparative HPLC. Compounds were screened in-vitro for their ability to activate the ARE on primary mouse cortical cultures as well as for their toxicity towards the cultures. All sesquiterpene lactones containing the α-methylene-γ-lactone moiety were able to activate the ARE although a number of compounds displayed significant cellular toxicity towards the cultures. The structure activity relationship of the sesquiterpene lactones indicate that the guaianolides isolated were more active and less toxic then the germacranolides. PMID:22923197

  5. A primary human macrophage-enteroid co-culture model to investigate mucosal gut physiology and host-pathogen interactions

    PubMed Central

    Noel, Gaelle; Baetz, Nicholas W.; Staab, Janet F.; Donowitz, Mark; Kovbasnjuk, Olga; Pasetti, Marcela F.; Zachos, Nicholas C.

    2017-01-01

    Integration of the intestinal epithelium and the mucosal immune system is critical for gut homeostasis. The intestinal epithelium is a functional barrier that secludes luminal content, senses changes in the gut microenvironment, and releases immune regulators that signal underlying immune cells. However, interactions between epithelial and innate immune cells to maintain barrier integrity and prevent infection are complex and poorly understood. We developed and characterized a primary human macrophage-enteroid co-culture model for in-depth studies of epithelial and macrophage interactions. Human intestinal stem cell-derived enteroid monolayers co-cultured with human monocyte-derived macrophages were used to evaluate barrier function, cytokine secretion, and protein expression under basal conditions and following bacterial infection. Macrophages enhanced barrier function and maturity of enteroid monolayers as indicated by increased transepithelial electrical resistance and cell height. Communication between the epithelium and macrophages was demonstrated through morphological changes and cytokine production. Intraepithelial macrophage projections, efficient phagocytosis, and stabilized enteroid barrier function revealed a coordinated response to enterotoxigenic and enteropathogenic E. coli infections. In summary, we have established the first primary human macrophage-enteroid co-culture system, defined conditions that allow for a practical and reproducible culture model, and demonstrated its suitability to study gut physiology and host responses to enteric pathogens. PMID:28345602

  6. Establishment of a primary hepatocyte culture from the small Indian mongoose (Herpestes auropunctatus) and distribution of mercury in liver tissue.

    PubMed

    Horai, Sawako; Yanagi, Kumiko; Kaname, Tadashi; Yamamoto, Masatatsu; Watanabe, Izumi; Ogura, Go; Abe, Shintaro; Tanabe, Shinsuke; Furukawa, Tatsuhiko

    2014-11-01

    The present study established a primary hepatocyte culture for the small Indian mongoose (Herpestes auropunctatus). To determine the suitable medium for growing the primary hepatic cells of this species, we compared the condition of cells cultured in three media that are frequently used for mammalian cell culture: Dulbecco's Modified Eagle's Medium, RPMI-1640, and William's E. Of these, William's E medium was best suited for culturing the hepatic cells of this species. Using periodic acid-Schiff staining and ultrastructural observations, we demonstrated the cells collected from mongoose livers were hepatocytes. To evaluate the distribution of mercury (Hg) in the liver tissue, we carried out autometallography staining. Most of the Hg compounds were found in the central region of hepatic lobules. Smooth endoplasmic reticulum, which plays a role inxenobiotic metabolism, lipid/cholesterol metabolism, and the digestion and detoxification of lipophilic substances is grown in this area. This suggested that Hg colocalized with smooth endoplasmic reticulum. The results of the present study could be useful to identify the detoxification systems of wildlife with high Hg content in the body, and to evaluate the susceptibility of wildlife to Hg toxicity.

  7. Propagation of human hepatitis A virus in African green monkey kidney cell culture: primary isolation and serial passage.

    PubMed Central

    Daemer, R J; Feinstone, S M; Gust, I D; Purcell, R H

    1981-01-01

    Human hepatitis A virus (HAV) was propagated in primary African Green Monkey (Cercopithecus aethiops) kidney (AGMK) cell cultures. Three strains of HAV were used: MS-1, SD-11, and HM-175. Cells were inoculated with marmoset-passaged material or human clinical specimens and were stained by direct immunofluorescence to establish the identity of the virus. Both clinical samples and marmoset-passaged material produced immunofluorescence. HAV antigen was found scattered throughout the cytoplasm of inoculated cultures. The HM-175 strain produced the most intense immunofluorescence. This strain of HAV had been serially passaged in cell culture seven times. Blocking experiments with paired human sera from naturally acquired HAV infections and hyperimmune chimpanzee serum from an experimentally infected animal established that the immunofluorescence was specific. The viral antigen was found to be exclusively intracellular. The interval to maximum HAV antigen expression was decreased by serial passage. The HAV strain described herein, which was recovered directly from the stool specimen of a patient with HAV in primary AGMK cell culture, may prove useful as a source of antigen for serological tests and as a candidate vaccine strain. Images PMID:6260685

  8. Propagation of human hepatitis A virus in African green monkey kidney cell culture: primary isolation and serial passage.

    PubMed

    Daemer, R J; Feinstone, S M; Gust, I D; Purcell, R H

    1981-04-01

    Human hepatitis A virus (HAV) was propagated in primary African Green Monkey (Cercopithecus aethiops) kidney (AGMK) cell cultures. Three strains of HAV were used: MS-1, SD-11, and HM-175. Cells were inoculated with marmoset-passaged material or human clinical specimens and were stained by direct immunofluorescence to establish the identity of the virus. Both clinical samples and marmoset-passaged material produced immunofluorescence. HAV antigen was found scattered throughout the cytoplasm of inoculated cultures. The HM-175 strain produced the most intense immunofluorescence. This strain of HAV had been serially passaged in cell culture seven times. Blocking experiments with paired human sera from naturally acquired HAV infections and hyperimmune chimpanzee serum from an experimentally infected animal established that the immunofluorescence was specific. The viral antigen was found to be exclusively intracellular. The interval to maximum HAV antigen expression was decreased by serial passage. The HAV strain described herein, which was recovered directly from the stool specimen of a patient with HAV in primary AGMK cell culture, may prove useful as a source of antigen for serological tests and as a candidate vaccine strain.

  9. Molecular responses to stress induced in normal human caucasian melanocytes in culture by exposure to simulated solar UV.

    PubMed

    Marrot, Laurent; Belaïdi, Jean-Philippe; Jones, Christophe; Perez, Philippe; Meunier, Jean-Roch

    2005-01-01

    Melanocytes play a central role in the response of skin to sunlight exposure. They are directly involved in UV-induced pigmentation as a defense mechanism. However, their alteration can lead to melanoma, a process where the role of sun overexposure is highly probable. The transformation process whereby UV damage may result in melanoma initiation is poorly understood, especially in terms of UV-induced genotoxicity in pigmented cells, where melanin can act either as a sunscreen or as a photosensitizer. The aim of this study was to analyze the behavior of melanocytes from fair skin under irradiation mimicking environmental sunlight in terms of spectral power distribution. To do this, normal human Caucasian melanocytes in culture were exposed to simulated solar UV (SSUV, 300-400 nm). Even at relatively high doses (until 20 min exposure, corresponding to 12 kJ/m2 UV-B and 110 kJ/m2 UV-A), cell death was limited, as shown by cell viability and low occurrence of apoptosis (caspase-3 activation). Moreover, p53 accumulation was three times lower in melanocytes than in unpigmented cells such as fibroblasts after SSUV exposure. However, an important fraction of melanocyte population was arrested in G2-M phase, and this correlated well with a high induction level of the gene GADD45, 4 h after exposure. Among the genes involved in DNA repair, gene XPC was the most inducible because its expression increased more than two-fold 15 h after a 20 min exposure, whereas expression of P48 was only slightly increased. In addition, an early induction of Heme Oxygenase 1 (HO1) gene, a typical response to oxidative stress, was also observed for the first time in melanocytes. Interestingly, this induction remained significant when melanocytes were exposed to UV-A radiation only (320-400 nm), and stimulation of melanogenesis before irradiation further increased HO1 induction. These results were obtained with normal human cells after exposure to SSUV radiation, which mimicked natural sunlight

  10. Maintenance of Hepatic Functions in Primary Human Hepatocytes Cultured on Xeno-Free and Chemical Defined Human Recombinant Laminins.

    PubMed

    Watanabe, Masaaki; Zemack, Helen; Johansson, Helene; Hagbard, Louise; Jorns, Carl; Li, Meng; Ellis, Ewa

    2016-01-01

    Refined methods for maintaining specific functions of isolated hepatocytes under xeno-free and chemical defined conditions is of great importance for the development of hepatocyte research and regenerative therapy. Laminins, a large family of heterotrimeric basement membrane adhesion proteins, are highly cell and tissue type specific components of the extracellular matrix and strongly influence the behavior and function of associated cells and/or tissues. However, detailed biological functions of many laminin isoforms are still to be evaluated. In this study, we determined the distribution of laminin isoforms in human liver tissue and isolated primary human hepatocytes by western blot analysis, and investigated the efficacy of different human recombinant laminin isoforms on hepatic functions during culture. Protein expressions of laminin-chain α2, α3, α4, β1, β3, γ1, and γ2 were detected in both isolated human hepatocytes and liver tissue. No α1 and α5 expression could be detected in liver tissue or hepatocytes. Hepatocytes were isolated from five different individual livers, and cultured on human recombinant laminin isoforms -111, -211, -221, -332, -411, -421, -511, and -521 (Biolamina AB), matrigel (extracted from Engelbreth-Holm-Swarm sarcoma), or collagen type IV (Collagen). Hepatocytes cultured on laminin showed characteristic hexagonal shape in a flat cell monolayer. Viability, double stranded DNA concentration, and Ki67 expression for hepatocytes cultured for six days on laminin were comparable to those cultured on EHS and Collagen. Hepatocytes cultured on laminin also displayed production of human albumin, alpha-1-antitrypsin, bile acids, and gene expression of liver-enriched factors, such as hepatocyte nuclear factor 4 alpha, glucose-6-phosphate, cytochrome P450 3A4, and multidrug resistance-associated protein 2. We conclude that all forms of human recombinant laminin tested maintain cell viability and liver-specific functions of primary human

  11. Perceptions of culturally competent diabetes management in a primary care practice.

    PubMed

    Kirk, Julienne K; Hildebrandt, Carol; Davis, Stephen; Crandall, Sonia J; Siciliano, Alissa B; Marion, Gail S

    2014-01-01

    To evaluate whether clinicians consider the impact of culture on diabetes management, a survey was mailed to 300 randomly selected patients > or = 50 years with type 2 diabetes and 153 surveys were returned. Data were correlated with A1C values. African Americans (AA) and non-Hispanic whites (NHW), (91.9%, 97.0%) respectively, reported clinicians discussed benefits of controlling blood sugar but did not discuss effects of cultural issues on glucose control (< or = 50%). AAs perceived clinicians were more accommodating of their cultural preferences than did NHWs (49.2% versus 30.6%) (P < .05). Females (51.9%) (P < .01) reported that clinicians acknowledged the importance of their cultural beliefs with a slightly higher percentage for African American females (54.8%) versus non-Hispanic White females (48.6%). Understanding the patient's and clinician's views of cultural beliefs as they relate to diabetes self-management can provide perspectives to guide care.

  12. Development of Methodology to Maintain Primary Cultures of Normal and Malignant Human Prostatic Epithelial Cells In Vivo

    DTIC Science & Technology

    2008-02-01

    grafting site. Several xenotransplantation models were developed based on severe combined immunodeficient (SCID) mice and their derivative, the non... xenotransplantation of human plasmacytoid dendritic cell precursors and human peripheral blood lymphocytes for the development of a severe acute graft-vs

  13. Development of Methodology to Maintain Primary Cultures of Normal and Malignant Human Prostatic Epihelial Cells in Vivo

    DTIC Science & Technology

    2007-02-01

    environment and the grafting site. Several xenotransplantation models were developed based on severe combined immunodeficient (SCID) mice and their...useful for studying xenotransplantation of human plasmacytoid dendritic cell precursors and human peripheral blood lymphocytes for the development of a

  14. Development of Methodology to Maintain Primary Cultures of Normal and Malignant Human Prostatic Epithelial Cells in Vivo

    DTIC Science & Technology

    2006-02-01

    xenotransplantation models were developed based on severe combined immunodeficient (SCID) mice and their derivative, the non-obese diabetic (NOD)/SCID mouse model. The...all T and B cells and NK function. This novel immunodeficient mouse proved to be useful for studying xenotransplantation of human plasmacytoid dendritic

  15. Isolation, purification, culture and characterisation of myoepithelial cells from normal and neoplastic canine mammary glands using a magnetic-activated cell sorting separation system.

    PubMed

    Sánchez-Céspedes, R; Maniscalco, L; Iussich, S; Martignani, E; Guil-Luna, S; De Maria, R; Martín de Las Mulas, J; Millán, Y

    2013-08-01

    Mammary gland tumours, the most common malignant neoplasm in bitches, often display myoepithelial (ME) cell proliferation. The aim of this study was to isolate, purify, culture and characterise ME cells from normal and neoplastic canine mammary glands. Monodispersed cells from three normal canine mammary glands and five canine mammary tumours were incubated with an anti-Thy1 antibody and isolated by magnetic-activated cell sorting (MACS). Cells isolated from two normal glands (cell lines CmME-N1 and CmME-N2) and four tumours (cell lines CmME-K1 from a complex carcinoma, CmME-K2 from a simple tubulopapillary carcinoma, and CmME-K3 and CmME-K4 from two carcinomas within benign tumours) were cultured in supplemented DMEM/F12 media for 40days. Cell purity was >90%. Tumour-derived ME cell lines exhibited heterogeneous morphology, growth patterns and immunocytochemical expression of cytokeratins, whereas cell lines from normal glands retained their morphology and levels of cytokeratin expression during culture. Cell lines from normal glands and carcinomas within benign tumours grew more slowly than those from simple and complex carcinomas. This methodology has the potential to be used for in vitro analysis of the role of ME cells in the growth and progression of canine mammary tumours.

  16. Ex vivo culture of primary human colonic tissue for studying transcriptional responses to 1α,25(OH)2 and 25(OH) vitamin D

    PubMed Central

    Mapes, Brandon; Chase, Meredith; Hong, Ellie; Ludvik, Anton; Ceryes, Katy; Huang, Yong

    2014-01-01

    1α,25-Dihydroxyvitamin D3 [1α,25(OH)2D3] is a steroid hormone derived from circulating 25(OH) vitamin D [25(OH)D] with chemopreventive effects in colorectal cancer. 1α,25(OH)2D3 acts through transcriptional mechanisms; however, our understanding of vitamin D transcriptional responses in the colon is derived from studies in transformed cancer cell lines which may not represent responses in normal healthy tissue. Here, we describe the optimization of an ex vivo culture model using primary colonic biopsy samples for studying short-term transcriptional response induced by 1α,25(OH)2D3 and 25(OH)D treatment. Colon biopsy samples from healthy subjects were maintained in primary culture and treated in parallel with 100 nM 1α,25(OH)2D3 or 62.5 nM 25(OH)D and vehicle control (ethanol). Viability was assessed using histology and enzymatic assays. Genome-wide transcriptional responses to 1α,25(OH)2D3 were assessed and expression of 25(OH)D targets CYP27B1 and CYP24A1 were measured by real time PCR. We show that ex vivo culture of colonic tissue remains viable for up to 8 h. The largest number of differentially expressed genes in response to 1α,25(OH)2D3 was noted after 6 h (n = 120). As proof of concept, the top upregulated gene was CYP24A1, a well-established vitamin D-responsive gene. With 25(OH)D treatment, mRNA expression of CYP27B1 was significantly increased after 1 h, while expression of CYP24A1 was greatest at 8 h. Ex vivo culture can be used to assess short-term transcriptional responses to 1α,25(OH)2D3 and 25(OH)D in primary tissue from human colon. Future studies will address interindividual differences in transcriptional responses. PMID:24550213

  17. Preparation and Culture of Myogenic Precursor Cells/Primary Myoblasts from Skeletal Muscle of Adult and Aged Humans.

    PubMed

    Soriano-Arroquia, Ana; Clegg, Peter D; Molloy, Andrew P; Goljanek-Whysall, Katarzyna

    2017-02-16

    Skeletal muscle homeostasis depends on muscle growth (hypertrophy), atrophy and regeneration. During ageing and in several diseases, muscle wasting occurs. Loss of muscle mass and function is associated with muscle fiber type atrophy, fiber type switching, defective muscle regeneration associated with dysfunction of satellite cells, muscle stem cells, and other pathophysiological processes. These changes are associated with changes in intracellular as well as local and systemic niches. In addition to most commonly used rodent models of muscle ageing, there is a need to study muscle homeostasis and wasting using human models, which due to ethical implications, consist predominantly of in vitro cultures. Despite the wide use of human Myogenic Progenitor Cells (MPCs) and primary myoblasts in myogenesis, there is limited data on using human primary myoblast and myotube cultures to study molecular mechanisms regulating different aspects of age-associated muscle wasting, aiding in the validation of mechanisms of ageing proposed in rodent muscle. The use of human MPCs, primary myoblasts and myotubes isolated from adult and aged people, provides a physiologically relevant model of molecular mechanisms of processes associated with muscle growth, atrophy and regeneration. Here we describe in detail a robust, inexpensive, reproducible and efficient protocol for the isolation and maintenance of human MPCs and their progeny - myoblasts and myotubes from human muscle samples using enzymatic digestion. Furthermore, we have determined the passage number at which primary myoblasts from adult and aged people undergo senescence in an in vitro culture. Finally, we show the ability to transfect these myoblasts and the ability to characterize their proliferative and differentiation capacity and propose their suitability for performing functional studies of molecular mechanisms of myogenesis and muscle wasting in vitro.

  18. Hormonally produced changes in caeruloplasmin synthesis and secretion in primary cultured rat hepatocytes. Relationship to hepatic copper metabolism.

    PubMed Central

    Weiner, A L; Cousins, R J

    1983-01-01

    Hormonally produced changes in the synthesis and secretion of the serum copper-containing protein caeruloplasmin were studied in primary cultures of rat liver parenchymal cells isolated by the collagenase-perfusion technique. A rabbit antibody directed against rat caeruloplasmin was used to immunoprecipitate labelled caeruloplasmin. Isolated liver cells synthesized and secreted caeruloplasmin over a period of 3 days. Synthesis and secretion of this protein was enhanced when cells were treated with dexamethasone. The accumulation of copper was also moderately enhanced with glucocorticoid treatment. Inclusion of adrenaline in the culture medium resulted in elevated incorporation of copper into newly synthesized caeruloplasmin as well as an increase in 64Cu-labelled caeruloplasmin in the culture medium. However, adrenaline did not seem to increase the secretion of 3H-labelled protein, despite the elevation in secreted 64Cu-caeruloplasmin. This may be due to a large increase in the intracellular pool of 64Cu caused by enhanced accumulation of this metal when adrenaline is included in the incubation medium. Enhanced copper accumulation was also seen when cells were treated with glucagon. Adrenaline-stimulated accumulation of 64Cu could be inhibited by including phenoxybenzamine, an alpha-adrenergic blocker, in the culture medium. Elevation of extracellular copper caused enhancement in the detection of labelled caeruloplasmin in the medium of cultured cells, probably owing to the ability of this metal to stabilize the protein. Images Fig. 1. PMID:6882374

  19. Surgical extraction of human dorsal root ganglia from organ donors and preparation of primary sensory neuron cultures

    PubMed Central

    Valtcheva, Manouela V.; Copits, Bryan A.; Davidson, Steve; Sheahan, Tayler D.; Pullen, Melanie Y.; McCall, Jordan G.; Dikranian, Krikor; Gereau, Robert W.

    2016-01-01

    Primary cultures of rodent sensory neurons are widely used to investigate the cellular and molecular mechanisms involved in pain, itch, nerve injury, and regeneration. However, translation of these preclinical findings may be greatly improved by direct validation in human tissues. We have developed an approach to extract and culture human sensory neurons in collaboration with a local organ procurement organization. Here we describe the surgical procedure for extraction of human dorsal root ganglia (hDRG) and the necessary modifications to existing culture techniques to prepare viable adult human sensory neurons for functional studies. Dissociated sensory neurons can be maintained in culture for >10 days, and are amenable to electrophysiological recording, calcium imaging, and viral gene transfer. The entire process of extraction and culturing can be completed in less than 7 hours, and can be performed by trained graduate students. This approach can be applied at any institution with access to organ donors consenting to tissue donation for research and provides an invaluable resource for improving translational research. PMID:27606776

  20. Ionizing Irradiation Not Only Inactivates Clonogenic Potential in Primary Normal Human Diploid Lens Epithelial Cells but Also Stimulates Cell Proliferation in a Subset of This Population

    PubMed Central

    Fujimichi, Yuki; Hamada, Nobuyuki

    2014-01-01

    Over the past century, ionizing radiation has been known to induce cataracts in the crystalline lens of the eye, but its mechanistic underpinnings remain incompletely understood. This study is the first to report the clonogenic survival of irradiated primary normal human lens epithelial cells and stimulation of its proliferation. Here we used two primary normal human cell strains: HLEC1 lens epithelial cells and WI-38 lung fibroblasts. Both strains were diploid, and a replicative lifespan was shorter in HLEC1 cells. The colony formation assay demonstrated that the clonogenic survival of both strains decreases similarly with increasing doses of X-rays. A difference in the survival between two strains was actually insignificant, although HLEC1 cells had the lower plating efficiency. This indicates that the same dose inactivates the same fraction of clonogenic cells in both strains. Intriguingly, irradiation enlarged the size of clonogenic colonies arising from HLEC1 cells in marked contrast to those from WI-38 cells. Such enhanced proliferation of clonogenic HLEC1 cells was significant at ≥2 Gy, and manifested as increments of ≤2.6 population doublings besides sham-irradiated controls. These results suggest that irradiation of HLEC1 cells not only inactivates clonogenic potential but also stimulates proliferation of surviving uniactivated clonogenic cells. Given that the lens is a closed system, the stimulated proliferation of lens epithelial cells may not be a homeostatic mechanism to compensate for their cell loss, but rather should be regarded as abnormal. This is because these findings are consistent with the early in vivo evidence documenting that irradiation induces excessive proliferation of rabbit lens epithelial cells and that suppression of lens epithelial cell divisions inhibits radiation cataractogenesis in frogs and rats. Thus, our in vitro model will be useful to evaluate the excessive proliferation of primary normal human lens epithelial cells that

  1. Additivity of Pyrethroid Actions on Sodium Influx in Cortical Neurons in Cerebrocortical Neurons in Primary Culture

    EPA Science Inventory

    BACKGROUND: Pyrethroid insecticides bind to voltage-gated sodium channels and modify their gating kinetics, thereby disrupting neuronal function. Although previous work has tested the additivity of pyrethroids in vivo, this has not been assessed directly at the primary molecular ...

  2. [Influence of different gelatin concentration and lymphocyte isolation liquid on primary culture of umbilical cord blood derived adhesive cells].

    PubMed

    Zhang, Cheng; Chen, Xing-Hua; Zhang, Xi; Gao, Lei; Kong, Pei-Yan; Liu, Hong; Liang, Xue; Peng, Xian-Gui; Wang, Qing-Yu

    2008-12-01

    In order to study the influence of different gelatin concentrations, and lymphocyte isolation liquid on primary culture of umbilical cord blood-derived adhesive cells (hCBACs), the red blood cells of umbilical cord blood was separated by 3% and 6 % gelatin for detecting the effectiveness of sedimentation, then the adhesion rate at 48 hours, the day of initial expansion and the rate of culture success were detected for hCBACs cultured with CD34(+) cells after the mononuclear cells were separated by 6% gelatin followed by Ficoll and Percoll, and the morphological characteristics and growth status were observed by invert microscopy. Cytochemistry stain for nonspecific esterase stain (NSE), peroxidase (POX), periodic acid Schiff reaction (PAS) and alkali phosphatase (ALP) and immunocytochemistry labeling for CD31, CD45, CD68 and fibronectin (Fn) were detected. The results showed that 6 % gelatin was better than that 3% gelatin for red blood sedimentation. The Percoll was predominant over Ficoll in adhesion rate at 48 hours, the day of initial expansion, the time of initial formation of adhesive cell colony units, the time of maximal numbers of adhesive cell colony units, the the cell fusion time and ratio of culture success. 60% fibroblast-liked cells, 36% macrophage liked cells and 4% small-round cells were observed in cells isolated by both isolated methods. The cytochemistry stain for NSE, POX, PAS and ALP was similar in two groups, the difference was not statistically significant between these two groups. The immunocytochemistry labeling for CD31, CD45, CD68 and Fn was also similar in both groups and the difference was also not statistically significant between these two groups. It is concluded that the combination of 6% gelatin with Percoll is an ideal separation method for primary culture of hCBACs, which provides basic information for clinical application.

  3. Adverse events analysis as an educational tool to improve patient safety culture in primary care: A randomized trial

    PubMed Central

    2011-01-01

    Background Patient safety is a leading item on the policy agenda of both major international health organizations and advanced countries generally. The quantitative description of the phenomena has given rise to intense concern with the issue in institutions and organizations, leading to a number of initiatives and research projects and the promotion of patient safety culture, with training becoming a priority both in Spain and internationally. To date, most studies have been conducted in a hospital setting, even though primary care is the type most commonly used by the public, in our experience. Our study aims to achieve the following: - Assess the registry of adverse events as an education tool to improve patient safety culture in the Family and Community Teaching Units of Galicia. - Find and analyze educational tools to improve patient safety culture in primary care. - Evaluate the applicability of the Hospital Survey on Patient Safety Culture by the Agency for Healthcare Research and Quality, Spanish version, in the context of primary health care. Design and methods Design Experimental unifactorial study of two groups, control and intervention. Study population Tutors and residents in Family and Community Medicine in last year of studies in Galicia, Spain. Sample From the population universe through voluntary participation. Twenty-seven tutor-resident units in each group required, randomly assigned. Intervention Residents and their respective tutor (tutor-resident pair) in teaching units on Family and Community Medicine from throughout Galicia will be invited to participate. Tutor-resident pair that agrees to participate will be sent the Hospital Survey on Patient Safety Culture. Then, tutor-resident pair will be assigned to each group-either intervention or control-through simple random sampling. The intervention group will receive specific training to record the adverse effects found in patients under their care, with subsequent feedback, after receiving

  4. High CD49f expression is associated with osteosarcoma tumor progression: a study using patient-derived primary cell cultures

    PubMed Central

    Penfornis, Patrice; Cai, David Z; Harris, Michael R; Walker, Ryan; Licini, David; Fernandes, Joseph D A; Orr, Griffin; Koganti, Tejaswi; Hicks, Chindo; Induru, Spandana; Meyer, Mark S; Khokha, Rama; Barr, Jennifer; Pochampally, Radhika R

    2014-01-01

    Overall prognosis for osteosarcoma (OS) is poor despite aggressive treatment options. Limited access to primary tumors, technical challenges in processing OS tissues, and the lack of well-characterized primary cell cultures has hindered our ability to fully understand the properties of OS tumor initiation and progression. In this study, we have isolated and characterized cell cultures derived from four central high-grade human OS samples. Furthermore, we used the cell cultures to study the role of CD49f in OS progression. Recent studies have implicated CD49f in stemness and multipotency of both cancer stem cells and mesenchymal stem cells. Therefore, we investigated the role of CD49f in osteosarcomagenesis. First, single cell suspensions of tumor biopsies were subcultured and characterized for cell surface marker expression. Next, we characterized the growth and differentiation properties, sensitivity to chemotherapy drugs, and anchorage-independent growth. Xenograft assays showed that cell populations expressing CD49fhi/CD90lo cell phenotype produced an aggressive tumor. Multiple lines of evidence demonstrated that inhibiting CD49f decreased the tumor-forming ability. Furthermore, the CD49fhi/CD90lo cell population is generating more aggressive OS tumor growth and indicating this cell surface marker could be a potential candidate for the isolation of an aggressive cell type in OSs. PMID:24802970

  5. Steroid metabolism by purified adult rat Leydig cells in primary culture

    SciTech Connect

    Browning, J.Y.; Tcholakian, R.K.; Kessler, M.J.; Grotjan, H.E. Jr.

    1982-11-01

    To characterize Leydig cell steroidogensis, we examined the metabolism of (3H)pregnenolone (3 beta-hydroxy-5-pregnen-20-one) to androgens in the presence and absence of human chorionic gonadotropin (hCG) as a function of culture duration. Approximately 20-30% of the (3H)pregnenolone was converted to testosterone (17 beta-hydroxy-4-androsten-3-one) by purified Leydig cells at 0, 3 and 5 days (d) of culture. Androstenedione (4-androstene-3,17-dione) and dihydrotestosterone (17 beta-hydroxy-5 alpha-androstan-3-one) were also produced while on day 5 of culture, significant amounts of progesterone (4-pregnene-3,20-dione) were isolated. The delta 5 intermediates, 17-hydroxypregnenolone (3 beta, 17-dihydroxy-5-pregnen-20-one) and dehydroepiandrosterone (3 beta-hydroxy-5-androsten-17-one), accounted for less than 1% of substrate conversion, indicating a clear preference for Leydig cells to metabolize (3H)pregnenolone via the delta 4 pathway. On day 0 of culture, unidentified metabolites considered of predominately polar steroids while on day 5 of culture, the unidentified metabolites consisted of predominately nonpolar steroids. In the presence of hCG, (3H-pregnenolone metabolism did not differ from basal on day 0 or 3 of culture. HCG increased the conversion of pregnenolone to progesterone and 17-hydroxyprogesterone (17-hydroxy-4-pregnene-3,20-dione) on 5d. This suggests that Leydig cells cultured for 5d have decreased C17-20 desmolase activity or that hCG acutely stimulates 3 beta-hydroxysteroid dehydrogenase and delta 5-delta 5 isomerase activities.

  6. Mild Heat Stress Enhances Angiogenesis in a Co-culture System Consisting of Primary Human Osteoblasts and Outgrowth Endothelial Cells

    PubMed Central

    Fuchs, Sabine; Böse, Thomas; Schmidt, Harald; Hofmann, Alexander; Tonak, Marcus; Unger, Ronald

    2014-01-01

    The repair and regeneration of large bone defects, including the formation of functional vasculature, represents a highly challenging task for tissue engineering and regenerative medicine. Recent studies have shown that vascularization and ossification can be stimulated by mild heat stress (MHS), which would offer the option to enhance the bone regeneration process by relatively simple means. However, the mechanisms of MHS-enhanced angiogenesis and osteogenesis, as well as potential risks for the treated cells are unclear. We have investigated the direct effect of MHS on angiogenesis and osteogenesis in a co-culture system of human outgrowth endothelial cells (OECs) and primary osteoblasts (pOBs), and assessed cytotoxic effects, as well as the levels of various heat shock proteins (HSPs) synthesized under these conditions. Enhanced formation of microvessel-like structures was observed in co-cultures exposed to MHS (41°C, 1 h), twice per week, over a time period of 7–14 days. As shown by real-time polymerase chain reaction (PCR), the expression of vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2), and tumor necrosis factor-alpha was up-regulated in MHS-treated co-cultures 24 h post-treatment. At the protein level, significantly elevated VEGF and Ang-1 concentrations were observed in MHS-treated co-cultures and pOB mono-cultures compared with controls, indicating paracrine effects associated with MHS-induced angiogenesis. MHS-stimulated co-cultures and OEC mono-cultures released higher levels of Ang-2 than untreated cultures. On the other hand MHS treatment of co-cultures did not result in a clear effect regarding osteogenesis. Nevertheless, real-time PCR demonstrated that MHS increased the expression of mitogen-activated protein kinase, interleukin-6, and bone morphogenetic protein 2, known as HSP-related molecules in angiogenic and osteogenic regulation pathways. In agreement with these observations, the expression of

  7. Mild heat stress enhances angiogenesis in a co-culture system consisting of primary human osteoblasts and outgrowth endothelial cells.

    PubMed

    Li, Ming; Fuchs, Sabine; Böse, Thomas; Schmidt, Harald; Hofmann, Alexander; Tonak, Marcus; Unger, Ronald; Kirkpatrick, Charles James

    2014-04-01

    The repair and regeneration of large bone defects, including the formation of functional vasculature, represents a highly challenging task for tissue engineering and regenerative medicine. Recent studies have shown that vascularization and ossification can be stimulated by mild heat stress (MHS), which would offer the option to enhance the bone regeneration process by relatively simple means. However, the mechanisms of MHS-enhanced angiogenesis and osteogenesis, as well as potential risks for the treated cells are unclear. We have investigated the direct effect of MHS on angiogenesis and osteogenesis in a co-culture system of human outgrowth endothelial cells (OECs) and primary osteoblasts (pOBs), and assessed cytotoxic effects, as well as the levels of various heat shock proteins (HSPs) synthesized under these conditions. Enhanced formation of microvessel-like structures was observed in co-cultures exposed to MHS (41°C, 1 h), twice per week, over a time period of 7-14 days. As shown by real-time polymerase chain reaction (PCR), the expression of vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2), and tumor necrosis factor-alpha was up-regulated in MHS-treated co-cultures 24 h post-treatment. At the protein level, significantly elevated VEGF and Ang-1 concentrations were observed in MHS-treated co-cultures and pOB mono-cultures compared with controls, indicating paracrine effects associated with MHS-induced angiogenesis. MHS-stimulated co-cultures and OEC mono-cultures released higher levels of Ang-2 than untreated cultures. On the other hand MHS treatment of co-cultures did not result in a clear effect regarding osteogenesis. Nevertheless, real-time PCR demonstrated that MHS increased the expression of mitogen-activated protein kinase, interleukin-6, and bone morphogenetic protein 2, known as HSP-related molecules in angiogenic and osteogenic regulation pathways. In agreement with these observations, the expression of some

  8. Low cost production of 3D-printed devices and electrostimulation chambers for the culture of primary neurons.

    PubMed

    Wardyn, Joanna D; Sanderson, Chris; Swan, Laura E; Stagi, Massimiliano

    2015-08-15

    The analysis of primary neurons is a basic requirement for many areas of neurobiology. However, the range of commercial systems available for culturing primary neurons is functionally limiting, and the expense of these devices is a barrier to both exploratory and large-scale studies. This is especially relevant as primary neurons often require unusual geometries and specialised coatings for optimum growth. Fortunately, the recent revolution in 3D printing offers the possibility to generate customised devices, which can support neuronal growth and constrain neurons in defined paths, thereby enabling many aspects of neuronal physiology to be studied with relative ease. In this article, we provide a detailed description of the system hardware and software required to produce affordable 3D-printed culture devices, which are also compatible with live-cell imaging. In addition, we also describe how to use these devices to grow and stimulate neurons within geometrically constrained compartments and provide examples to illustrate the practical utility and potential that these protocols offer for many aspects of experimental neurobiology.

  9. Low cost production of 3D-printed devices and electrostimulation chambers for the culture of primary neurons

    PubMed Central

    Wardyn, Joanna D.; Sanderson, Chris; Swan, Laura E.; Stagi, Massimiliano

    2015-01-01

    The analysis of primary neurons is a basic requirement for many areas of neurobiology. However, the range of commercial systems available for culturing primary neurons is functionally limiting, and the expense of these devices is a barrier to both exploratory and large-scale studies. This is especially relevant as primary neurons often require unusual geometries and specialised coatings for optimum growth. Fortunately, the recent revolution in 3D printing offers the possibility to generate customised devices, which can support neuronal growth and constrain neurons in defined paths, thereby enabling many aspects of neuronal physiology to be studied with relative ease. In this article, we provide a detailed description of the system hardware and software required to produce affordable 3D-printed culture devices, which are also compatible with live-cell imaging. In addition, we also describe how to use these devices to grow and stimulate neurons within geometrically constrained compartments and provide examples to illustrate the practical utility and potential that these protocols offer for many aspects of experimental neurobiology. PMID:25962333

  10. Cinacalcet hydrochloride in combination with alendronate normalizes hypercalcemia and improves bone mineral density in patients with primary hyperparathyroidism.

    PubMed

    Faggiano, A; Di Somma, C; Ramundo, V; Severino, R; Vuolo, L; Coppola, A; Panico, F; Savastano, S; Lombardi, G; Colao, A; Gasperi, M

    2011-06-01

    Cinacalcet is effective in controlling the biochemical abnormalities in patients with primary hyperparathyroidism (PHPT) but it seems to be less effective on bone mineral density (BMD). In the same patients, bisphosphonates are reported to be effective on bone resorption but less effective on calcium and PTH excess. In this study, the efficacy of cinacalcet in combination with alendronate has been retrospectively evaluated in patients with PHPT. Twenty-three patients with PHPT who had not been operated were retrospectively investigated. Cinacalcet was evaluated in combination with alendronate in 10 of the 23 patients, and in monotherapy in 13 other patients. Serum calcium, phosphorus and PTH, 24 h urine calcium and phosphorus as well as BMD, evaluated by DXA and expressed as T-score, were measured before and after treatment. In all patients serum calcium and phosphorus and urinary calcium excretion were effectively and stably controlled and PTH was significantly decreased after treatment. There was no difference in the rate of serum calcium and PTH decrease between subjects treated with cinacalcet plus alendronate and those treated with cinacalcet alone. T-score increased by 9.6% at lumbar spine and 3.9% at femur level in the cinacalcet plus alendronate subgroup and was unchanged in the cinacalcet subgroup (P < 0.01). In patients with PHPT, the biochemical abnormalities are rapidly improved by cinacalcet regardless from the administration in monotherapy or in combination with alendronate. BMD is significantly improved in patients receiving cinacalcet plus alendronate and stable in those receiving cinacalcet in monotherapy.

  11. Ultra-structural effects of different low-level lasers on normal cultured human melanocytes: an in vitro comparative study.

    PubMed

    AlGhamdi, Khalid M; Kumar, Ashok; A Al-Ghamdi, Attieh; Al-Rikabi, Ammar C; Mubarek, Mohammed; Ashour, Abdelkader E

    2016-12-01

    The aim of this study was to investigate the effects of the different types of low-level laser therapy (LLLT) on the ultra-structure and number of melanosomes in normal cultured human melanocytes. Specific effects of various types of LLLT on the ultra-structure of melanosomes have not yet been reported. Melanocytes were exposed to LLLT at an energy level of 2.0 J/cm(2), using a blue (457 nm), red (635 nm), or ultraviolet (UV) (355 nm) laser. After 72 h of irradiation, the melanocytes were fixed in 2.5 % glutaraldehyde (pH 7.2) phosphate buffer for 8 h and analyzed by transmission electron microscopy. Four developmental stages (I to IV) of melanosomes were observed, and their numbers were counted manually. The percentage of stages I, II, III, and IV melanosomes was 12.8, 14.2, 22.6, and 50.3 %, respectively, in the control (sham light). However, the melanosome percentages were 41.2, 5.4, 8.2, and 24.2 % in stages I, II, III, and IV, respectively, in the blue laser-treated group; 58.4, 6.1, 9.3, and 26.2 % for stages I, II, III, and IV, respectively, in the red laser-treated group; and 31.3, 11.1, 16.5, and 41.1 % for stages I, II, III, and IV, respectively, in the UV laser-treated group. The present data show that the amount of stage I is significantly higher (P < 0.0001) in the LLLT-treated cells compared to the control, which indicates significant stimulation of melanogenesis. The red laser was more effective than the other lasers. Moreover, the effects of LLLT on the ultra-structure of melanosomes need to be studied in a larger number of subject groups.

  12. On-chip, multisite extracellular and intracellular recordings from primary cultured skeletal myotubes

    PubMed Central

    Rabieh, Noha; Ojovan, Silviya M.; Shmoel, Nava; Erez, Hadas; Maydan, Eilon; Spira, Micha E.

    2016-01-01

    In contrast to the extensive use of microelectrode array (MEA) technology in electrophysiological studies of cultured neurons and cardiac muscles, the vast field of skeletal muscle research has yet to adopt the technology. Here we demonstrate an empowering MEA technology for high quality, multisite, long-term electrophysiological recordings from cultured skeletal myotubes. Individual rat skeletal myotubes cultured on micrometer sized gold mushroom-shaped microelectrode (gMμE) based MEA tightly engulf the gMμEs, forming a high seal resistance between the myotubes and the gMμEs. As a consequence, spontaneous action potentials generated by the contracting myotubes are recorded as extracellular field potentials with amplitudes of up to 10 mV for over 14 days. Application of a 10 ms, 0.5–0.9 V voltage pulse through the gMμEs electroporated the myotube membrane, and transiently converted the extracellular to intracellular recording mode for 10–30 min. In a fraction of the cultures stable attenuated intracellular recordings were spontaneously produced. In these cases or after electroporation, subthreshold spontaneous potentials were also recorded. The introduction of the gMμE-MEA as a simple-to-use, high-quality electrophysiological tool together with the progress made in the use of cultured human myotubes opens up new venues for basic and clinical skeletal muscle research, preclinical drug screening, and personalized medicine. PMID:27812002

  13. Human embryonic stem cells in culture possess primary cilia with hedgehog signaling machinery.

    PubMed

    Kiprilov, Enko N; Awan, Aashir; Desprat, Romain; Velho, Michelle; Clement, Christian A; Byskov, Anne Grete; Andersen, Claus Y; Satir, Peter; Bouhassira, Eric E; Christensen, Søren T; Hirsch, Rhoda Elison

    2008-03-10

    Human embryonic stem cells (hESCs) are potential therapeutic tools and models of human development. With a growing interest in primary cilia in signal transduction pathways that are crucial for embryological development and tissue differentiation and interest in mechanisms regulating human hESC differentiation, demonstrating the existence of primary cilia and the localization of signaling components in undifferentiated hESCs establishes a mechanistic basis for the regulation of hESC differentiation. Using electron microscopy (EM), immunofluorescence, and confocal microscopies, we show that primary cilia are present in three undifferentiated hESC lines. EM reveals the characteristic 9 + 0 axoneme. The number and length of cilia increase after serum starvation. Important components of the hedgehog (Hh) pathway, including smoothened, patched 1 (Ptc1), and Gli1 and 2, are present in the cilia. Stimulation of the pathway results in the concerted movement of Ptc1 out of, and smoothened into, the primary cilium as well as up-regulation of GLI1 and PTC1. These findings show that hESCs contain primary cilia associated with working Hh machinery.

  14. COMMUNICATION: Folate and S-adenosylmethionine modulate synaptic activity in cultured cortical neurons: acute differential impact on normal and apolipoprotein-deficient mice

    NASA Astrophysics Data System (ADS)

    Serra, Michael; Chan, Amy; Dubey, Maya; Gilman, Vladimir; Shea, Thomas B.

    2008-12-01

    Folate deficiency is accompanied by a decline in the cognitive neurotransmitter acetylcholine and a decline in cognitive performance in mice lacking apolipoprotein E (ApoE-/- mice), a low-density lipoprotein that regulates aspects of lipid metabolism. One direct consequence of folate deficiency is a decline in S-adenosylmethionine (SAM). Since dietary SAM supplementation maintains acetylcholine levels and cognitive performance in the absence of folate, we examined herein the impact of folate and SAM on neuronal synaptic activity. Embryonic cortical neurons from mice expressing or lacking ApoE (ApoE+/+ or -/-, respectively) were cultured for 1 month on multi-electrode arrays, and signaling was recorded. ApoE+/+ cultures displayed significantly more frequent spontaneous signals than ApoE-/- cultures. Supplementation with 166 µm SAM (not normally present in culture medium) increased signal frequency and decreased signal amplitude in ApoE+/+ cultures. SAM also increased the frequency of tightly clustered signal bursts. Folate deprivation reversibly reduced signal frequency in ApoE+/+ cultures; SAM supplementation maintained signal frequency despite folate deprivation. These findings support the importance of dietary supplementation with folate and SAM on neuronal health. Supplementation with 166 µm SAM did not alter signaling in ApoE-/- cultures, which may be a reflection of the reduced SAM levels in ApoE-/- mice. The differential impact of SAM on ApoE+/+ and -/- neurons underscores the combined impact of nutritional and genetic deficiencies on neuronal homeostasis.

  15. WORKING CONFERENCE ON RESEARCH AND ACTIVITY IN THE LANGUAGE ARTS FOR THE PRE-PRIMARY/PRIMARY CULTURALLY DIVERSE NON-ENGLISH SPEAKING CHILD (ALBUQUERQUE, JUNE 4-6, 1967).

    ERIC Educational Resources Information Center

    SMOKER, DAVID

    A WORKING CONFERENCE, SPONSORED BY THE SOUTHWESTERN COOPERATIVE EDUCATIONAL LABORATORY, INC., WAS HELD IN ALBUQUERQUE, NEW MEXICO ON JUNE 4-6, 1967 TO BRING TOGETHER PERSONS INTERESTED IN RELATED RESEARCH ACTIVITIES IN THE AREA OF LANGUAGE ARTS FOR PRE-PRIMARY AND PRIMARY, CULTURALLY DIVERSE, NON-ENGLISH SPEAKING CHILDREN. PARTICIPANTS INVITED…

  16. [Culture and health services: studying the participation of cultural traits of Brazilian society in the work process of primary healthcare services].

    PubMed

    Pinto, Alessandra Maria Silva; Najar, Alberto Lopes

    2011-11-01

    The analysis of institutions is a widely researched area of health. The culture of organizations is understood as a symbolic possibility contained in a larger dimension, called "national culture". This premise justifies the incorporation of the social anthropological approach to the study of organizational culture. This study sought to establish the perceptions of employees of two primary healthcare services in Niterói, State of Rio de Janeiro, regarding commonly used social navigation strategies from the theory developed by Roberto DaMatta. The results showed the relational character associated with the stereotype of the Brazilian people manifested by conflicts arising from the existence of values based on the `individual' and the `person'. Among them are the distortions observed between discourse and practice, and the mobilization strategies of social navigation like "making do" - to establish a mediation between the person and the impersonal law. The organization of the services of the Niterói Family Medical Program apparently sets its employees the concrete challenge of balancing the egalitarian principle that underpins the Unified Health System (SSU) with the set of values upon which personal relations are based in Brazilian society.

  17. Primary cultures of corticostriatal cells from newborn rats: a model to study muscarinic receptor subtypes regulation and function.

    PubMed

    Eva, C; Bovolin, P; Balzac, F; Botta, C; Gamalero, S R; Vaccarino, F M

    1990-01-01

    In the present work we characterized both the presynaptic and postsynaptic components of cholinergic transmission in a primary culture of corticostriatal neurons prepared from newborn rat brain. This culture preparation contains a small population of choline acetyltransferase (ChAT) immunoreactive neurons, corresponding to approximately 3% of the total cell number, and synthesizes increasing amounts of acetylcholine (ACh) from the third day in vitro (DIV), which reaches a plateau around the 10 day of culture. Muscarinic cholinergic receptors (mAChR), measured by the binding of the muscarinic antagonist [3H]quinuclidinyl benzilate ([3H]QNB), are detectable from the fifth DIV and increase linearly during the time of culture. At the twelfth DIV, the density of mAChRs (approximately 600 fmol/mg protein) is comparable to the density of mAChR in adult rat cortex. These receptors are coupled to second messenger systems, since muscarinic agonists inhibit adenylate cyclase activity and stimulate phosphoinositide breakdown with efficacies and potencies similar to those found in adult rat cortex. Moreover, by using the reverse transcriptase-polymerase chain reaction (RT-PCR) technique, we were able to demonstrate the presence of the m1, m3, and m4 mAChR subtype mRNAs in this neuronal culture at 12 DIV. Our data suggest that corticostriatal neuronal cultures develop in vitro ACh-synthesizing neurons and functionally active cholinergic receptors. This therefore makes them ideally suited to study the development and properties of brain mAChR subtypes.

  18. Ganoderma Lucidum polysaccharides protect against MPP+ and rotenone-induced apoptosis in primary dopaminergic cell cultures through inhibiting oxidative stress

    PubMed Central

    Guo, Shan-Shan; Cui, Xiao-Lan; Rausch, Wolf-Dieter

    2016-01-01

    Oxidative stress plays a pivotal role in the progressive neurodegeneration in Parkinson’s disease (PD) which is responsible for disabling motor abnormalities in more than 6.5 million people worldwide. Polysaccharides are the main active constituents from Ganoderma lucidum which is characterized with anti-oxidant, antitumor and immunostimulant properties. In the present study, primary dopaminergic cell cultures prepared from embryonic mouse mesencephala were used to investigate the neuroprotective effects and the potential mechanisms of Ganoderma lucidum polysaccharides (GLP) on the degeneration of dopaminergic neurons induced by the neurotoxins methyl-4-phenylpyridine (MPP+) and rotenone. Results revealed that GLP can protect dopamine neurons against MPP+ and rotenone at the concentrations of 100, 50 and 25 μg/ml in primary mesencephalic cultures in a dose-dependent manner. Interestingly, either with or without neurotoxin treatment, GLP treatment elevated the survival of THir neurons, and increased the length of neurites of dopaminergic neurons. The Trolox equivalent anti-oxidant capacity (TEAC) of GLP was determined to be 199.53 μmol Trolox/g extract, and the decrease of mitochondrial complex I activity induced by MPP+ and rotenone was elevated by GLP treatment (100, 50, 25 and 12.5 μg/ml) in a dose dependent manner. Furthermore, GLP dramatically decreased the relative number of apoptotic cells and increased the declining mitochondrial membrane potential (ΔΨm) induced by MPP+ and rotenone in a dose-dependent manner. In addition, GLP treatment reduced the ROS formation induced by MPP+ and rotenone at the concentrations of 100, 50 and 25 μg/ml in a dose-dependent manner. Our study indicates that GLP possesses neuroprotective properties against MPP+ and rotenone neurotoxicity through suppressing oxidative stress in primary mesencephalic dopaminergic cell culture owning to its antioxidant activities. PMID:27335703

  19. Epoxypukalide Induces Proliferation and Protects against Cytokine-Mediated Apoptosis in Primary Cultures of Pancreatic β-Cells

    PubMed Central

    López-Acosta, José Francisco; Moreno-Amador, José Luis; Jiménez-Palomares, Margarita; Díaz-Marrero, Ana R.; Cueto, Mercedes; Perdomo, Germán; Cózar-Castellano, Irene

    2013-01-01

    There is an urgency to find new treatments for the devastating epidemic of diabetes. Pancreatic β-cells viability and function are impaired in the two most common forms of diabetes, type 1 and type 2. Regeneration of pancreatic β-cells has been proposed as a potential therapy for diabetes. In a preliminary study, we screened a collection of marine products for β-cell proliferation. One unique compound (epoxypukalide) showed capability to induce β-cell replication in the cell line INS1 832/13 and in primary rat cell cultures. Epoxypukalide was used to study β-cell proliferation by [3H]thymidine incorporation and BrdU incorporation followed by BrdU/insulin staining in primary cultures of rat islets. AKT and ERK1/2 signalling pathways were analyzed. Cell cycle activators, cyclin D2 and cyclin E, were detected by western-blot. Apoptosis was studied by TUNEL and cleaved caspase 3. β-cell function was measured by glucose-stimulated insulin secretion. Epoxypukalide induced 2.5-fold increase in β-cell proliferation; this effect was mediated by activation of ERK1/2 signalling pathway and upregulation of the cell cycle activators, cyclin D2 and cyclin E. Interestingly, epoxypukalide showed protection from basal (40% lower versus control) and cytokine-induced apoptosis (80% lower versus control). Finally, epoxypukalide did not impair β-cell function when measured by glucose-stimulated insulin secretion. In conclusion, epoxypukalide induces β-cell proliferation and protects against basal and cytokine-mediated β-cell death in primary cultures of rat islets. These findings may be translated into new treatments for diabetes. PMID:23300997

  20. Testing the committee approach to translating measures across cultures: Translating primary communication inventory from English to Japanese.

    PubMed

    Furukawa, Ryoko; Driessnack, Martha

    2016-12-01

    This paper presents the initial translation process and follow-up psychometric evaluation of the Japanese version of the Primary Communication Inventory (J-PCI). The J-PCI was developed using the committee approach to translation and then used in a study exploring Japanese couples' communication satisfaction while separated during Satogaeri Bunben - a Japanese perinatal tradition. The committee approach attends to cultural nuance and context and is especially useful when languages have dissimilar linguistic roots and cultures, such as Japanese and English. The translation process and evaluation included five steps; (i) selection of the original PCI for research; (ii) selection of translators; (iii) development of the J-PCI using a committee approach; (iv) an initial small pilot study; and (v) a larger follow-up study. The J-PCI has good initial validity and reliability, although some nuances were observed in scoring.

  1. Microscopy, culture, and sensitive management of uncomplicated urinary tract infections in adults in the primary care setting.

    PubMed

    Sivathasan, Niroshan; Rakowski, Krzysztof R

    2011-06-01

    The high prevalence of urinary tract infections (UTIs) places a significant burden on healthcare systems. Clinicians may over-manage the issue, and there is great variability in practice, with economic- and resource- implications. Up to 40% of patients with a suspected UTI do not have an infection. Using PubMed (Medline) to shortlist relevant papers in English from the last 30 years, and further sub-selection to include only uncomplicated UTIs in adults in primary care, we reviewed the literature pertaining to uncomplicated UTIs, and how it should be managed efficiently in the primary care setting. In general practice, there is no advantage to routinely request microscopy and culture of urine samples in the presence of an appropriate history and urinalysis reagent-strip testing. If antibiotics are required, then a 3-day course shall suffice. Larger epidemiological studies focusing on more susceptible sub-populations may provide better guidance for discriminatory factors to produce an algorithm for treatment.

  2. Use of primary cell cultures to measure the late effects in the skins of rhesus monkeys irradiated with protons

    NASA Astrophysics Data System (ADS)

    Cox, A. B.; Wood, D. H.; Lett, J. T.

    Previous pilot investigations of the uses of primary cell cultures to study late damage in stem cells of the skin of the New Zealand white (NZW) rabbit and the rhesus monkey /1-3/, have been extended to individual monkeys exposed to 55 MeV protons. Protons of this energy have a larger range in tissue of (~2.6 cm) than the 32 MeV protons (~0.9 cm) to which the animals in our earlier studies had been exposed. Although the primary emphases in the current studies were improvement and simplification in the techniques and logistics of transportation of biopsies to a central analytical facility, comparison of the quantitative measurements obtained thus far for survival of stem cells in the skins from animals irradiated 21 years ago reveals that the effects of both proton energies are similar.

  3. Data on acylglycerophosphate acyltransferase 4 (AGPAT4) during murine embryogenesis and in embryo-derived cultured primary neurons and glia

    PubMed Central

    Bradley, Ryan M.; Mardian, Emily B.; Marvyn, Phillip M.; Vasefi, Maryam S.; Beazely, Michael A.; Mielke, John G.; Duncan, Robin E.

    2015-01-01

    Whole mouse embryos at three developmental timepoints, embryonic (E) day E10.5, E14.5, and E18.5, were analyzed for Agpat4 mRNA expression. Primary cortical mouse cultures prepared from E18.5 mouse brains were used for immunohistochemistry. Our data show that Agpat4 is differentially expressed at three timepoints in murine embryogenesis and is immunodetectable in both neurons and glial cells derived from the developing mouse brain. This paper contains data related to research concurrently published in Bradley et al. (2015) [1]. PMID:26759825

  4. Tryptophan pyrolysis products, Trp-P-1 and Trp-P-2 induce apoptosis in primary cultured rat hepatocytes.

    PubMed

    Ashida, H; Shiotani, B; Adachi, H; Hashimoto, T; Kanazawa, K; Danno, G

    1998-11-01

    The cytotoxicity of heterocyclic amines, dietary carcinogens derived from cooked foods, to primary cultured rat hepatocytes was studied. A tryptophan pyrolysis product, 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) was the most cytotoxic of 11 compounds tested. Trp-P-1 was found to induce apoptosis as measured by morphological changes in nuclear chromatin and internucleosomal DNA fragmentation. 3-Amino-1-methyl-5H-pyrido[4,3-b] indole (Trp-P-2) showed a moderate apoptotic effect, and other compounds had a similar but weaker effect.

  5. Data on acylglycerophosphate acyltransferase 4 (AGPAT4) during murine embryogenesis and in embryo-derived cultured primary neurons and glia.

    PubMed

    Bradley, Ryan M; Mardian, Emily B; Marvyn, Phillip M; Vasefi, Maryam S; Beazely, Michael A; Mielke, John G; Duncan, Robin E

    2016-03-01

    Whole mouse embryos at three developmental timepoints, embryonic (E) day E10.5, E14.5, and E18.5, were analyzed for Agpat4 mRNA expression. Primary cortical mouse cultures prepared from E18.5 mouse brains were used for immunohistochemistry. Our data show that Agpat4 is differentially expressed at three timepoints in murine embryogenesis and is immunodetectable in both neurons and glial cells derived from the developing mouse brain. This paper contains data related to research concurrently published in Bradley et al. (2015) [1].

  6. Allelic loss of chromosome 8p22 loci in a primary tissue culture from a patient with prostate cancer

    SciTech Connect

    Godec, C.J.; Macera, M.J.; Szabo, P. |

    1994-09-01

    It is presently estimated that over 11 million men in the United States alone have histological prostate cancer. Approximately 200,000 new cases will be diagnosed in 1994 with 36,000 deaths attributed to the most common cancer in U.S. men. Although the incidence of this disease is so high, little is known about the molecular etiology of this cancer. Inactivation of tumor suppressor genes Rb and p53 have been observed at a low frequency in this disease, although allelic loss on chromosomes 8, 10 and 16 suggest possible unidentified tumor suppression genes may be more directly associated with this cancer. It was recently shown that allelic loss of D8S163, located at 8q22 {r_arrow} pter, was seen in a large percentage of primary prostate cancers. Our patient underwent a radical prostatectomy for clinically localized prostate cancer. A portion of the tissue was minced, treated with collagenase and a fibroblast-like culture was established. DNA was obtained from the culture and peripheral blood of the patient. Probe K5R2 (American Type Culture Collection) specific for loci D8S163 was hybridized to Southern blots of TagI-digested DNA. Two alleles at 3.3 Kb and 1.9 Kb were seen in the peripheral blood, while only the 1.9 Kb fragment was seen in the DNA from the culture. Loss of the allele at 3.3 Kb resembles results obtained from primary tumors, suggesting that the established line may be derived from the tumor.

  7. Real-life data on antibiotic prescription and sputum culture diagnostics in acute exacerbations of COPD in primary care

    PubMed Central

    Bathoorn, Erik; Groenhof, Feikje; Hendrix, Ron; van der Molen, Thys; Sinha, Bhanu; Kerstjens, Huib AM; Friedrich, Alex W; Kocks, Janwillem WH

    2017-01-01

    Background Acute exacerbations of chronic obstructive pulmonary disease (AECOPD) are generally treated with optimization of bronchodilation therapy and a course of oral corticosteroids, mostly without antibiotics. The Dutch guidelines recommend prudent use of antibiotics, with amoxicillin or doxycycline as first choice. Here we evaluate adherence to these guidelines with regard to antibiotic prescription in AECOPD in primary care and the use of sputum cultures. Methods We retrospectively analyzed a longitudinal cohort of patients in three primary care practices in the north-eastern region of the Netherlands from 2009 to 2013 (n=36,172 subjects) participating in the Registration Network Groningen. Antibiotics prescribed for AECOPD −10/+28 days from the start date of corticosteroid courses were evaluated. In addition, we assessed regional data on the susceptibility of respiratory pathogens from COPD patients. Results We identified 1,297 patients with COPD. Of these, 616 experienced one or more exacerbations, resulting in a total of 1,558 exacerbations, for which 1,594 antibiotic courses were prescribed. The recommended antibiotics doxycycline and amoxicillin accounted for 56% of the prescribed antibiotics overall and for 35% in subsequent antibiotic courses. The alternative choices were not based on culture results because only in 67 AECOPD events (2.9%) sputum samples were taken. Regional data including 3,638 sputum samples showed that pathogens relevant in AECOPD were detected in 19% of cultures. Conclusion Our study shows that guidelines regarding the prescription of antibiotics are poorly followed, particularly in recurrent exacerbations. Sputum cultures were performed in a small minority of cases. Performing sputum diagnostics in patients with early treatment failure or a repeated exacerbation when antibiotic treatment is started may further rationalize antibiotic treatment. PMID:28144133

  8. 5-HT7 Receptors Are Not Involved in Neuropeptide Release in Primary Cultured Rat Trigeminal Ganglion Neurons.

    PubMed

    Wang, Xiaojuan; Hu, Rong; Liang, Jianbo; Li, Ze; Sun, Weiwen; Pan, Xiaoping

    2016-06-01

    Migraine is a common but complex neurological disorder. Its precise mechanisms are not fully understood. Increasing indirect evidence indicates that 5-HT7 receptors may be involved; however, their role remains unknown. Our previous in vivo study showed that selective blockade of 5-HT7 receptors caused decreased serum levels of calcitonin gene-related peptide (CGRP) in the external jugular vein following electrical stimulation of the trigeminal ganglion (TG) in an animal model of migraine. In the present study, we used an in vitro model of cultured TG cells to further investigate whether 5-HT7 receptors are directly responsible for the release of CGRP and substance P from TG neurons. We stimulated rat primary cultured TG neurons with capsaicin or potassium chloride (KCl) to mimic neurogenic inflammation, resulting in release of CGRP and substance P. 5-HT7 receptors were abundantly expressed in TG neurons. Greater than 93 % of 5-HT7 receptor-positive neurons co-expressed CGRP and 56 % co-expressed substance P. Both the capsaicin- and KCl-induced release of CGRP and substance P were unaffected by pretreatment of cultured TG cells with the selective 5-HT7 receptor agonist AS19 and antagonist SB269970. This study demonstrates for the first time that 5-HT7 receptors are abundantly co-expressed with CGRP and substance P in rat primary TG neurons and suggests that they are not responsible for the release of CGRP and substance P from cultured TG neurons evoked by capsaicin or KCl.

  9. Iodine regulates G2/M progression induced by CCL21/CCR7 interaction in primary cultures of papillary thyroid cancer cells with RET/PTC expression.

    PubMed

    Zhang, You-Yuan; Liu, Ze-Bing; Ye, Xuan-Guang; Ren, Wei-Min

    2016-10-01

    Treatment with high iodine concentrations can delay oncogenic activation effects, reduce cell growth and return thyroid-specific gene and protein expression levels to normal. During rearranged during transfection (RET)/papillary thyroid carcinoma (PTC) 3 activation, excess iodine can act as a protective agent in thyroid follicular cells. The chemokine receptor CCR7 serves a critical role in lymphocyte trafficking into and within lymph nodes, the preferential metastatic site for PTC. However, the potential associations between chemokine (C‑C motif) ligand 21 (CCL21)/C‑C chemokine receptor type 7 (CCR7) interaction and iodine concentrations in primary cultures of PTC with RET/PTC expression remain unclear. Proliferation assays of primary cultures of PTC cells with RET/PTC1 and RET/PTC3 expression indicated that CCR7 activation by its specific ligand, CCL21, was associated with significantly increased cell proliferation. Flow cytometry data indicated that CCL21/CCR7 interaction significantly increased the fraction of cells in the G2/M phase of the cell cycle. Western blotting indicated that CCL21/CCR7 interaction significantly upregulated cyclin A, cyclin B1 and cyclin‑dependent kinase 1 (CDK1) expression. Western blotting determined that CCL21/CCR7 interaction significantly enhanced the levels of phosphorylated extracellular signal‑regulated kinase (P‑ERK). Co-immunoprecipitation confirmed that there was interaction between P‑ERK and cyclin A, cyclin B1 or CDK1, particularly in the presence of CCL21. Sodium iodide (NaI, 10-5 M) significantly abolished the effects of exogenous CCL21. These results suggest that CCL21/CCR7 interaction contributes to G2/M progression of RET/PTC‑expressing cells via the ERK pathway in association with 10‑5 M NaI.

  10. Primary-Grade Students' Knowledge and Thinking about Transportation as a Cultural Universal.

    ERIC Educational Resources Information Center

    Brophy, Jere; Alleman, Janet

    The traditional K-3 social studies curriculum has focused on food, clothing, shelter, communication, transportation, and other cultural universals. Very little information exists about children's prior knowledge and thinking about these topics. This study was designed to provide such information with respect to the topic of transportation, and in…

  11. Primary-Grade Students' Knowledge and Thinking about Communication as a Cultural Universal.

    ERIC Educational Resources Information Center

    Brophy, Jere; Alleman, Janet

    The traditional K-3 social studies curriculum has focused on food, clothing, shelter, communication, transportation, and other cultural universals. Little information exists about children's prior knowledge and thinking (including misconceptions) about these topics. This study was designed to provide such information with respect to the topic of…

  12. Primary-Grade Students' Knowledge and Thinking about Family Living as a Cultural Universal.

    ERIC Educational Resources Information Center

    Brophy, Jere; Alleman, Janet

    The traditional K-3 social studies curriculum has focused on food, clothing, shelter, communication, transportation, and other cultural universals. Very little information exists about children's prior knowledge and thinking about these topics. This study was designed to provide such information with respect to the topic of family living, and in…

  13. Primary-Grade Students' Knowledge and Thinking about Food as a Cultural Universal.

    ERIC Educational Resources Information Center

    Brophy, Jere; Alleman, Janet; O'Mahony, Carolyn

    The traditional K-3 social studies curriculum has focused on food, clothing, shelter, communication, transportation, and other cultural universals, but little information exists about children's prior knowledge and thinking (including misconceptions) about these topics. This study was designed to provide such information with respect to the topic…

  14. Flavonoids modulate the proliferation of Neospora caninum in glial cell primary cultures.

    PubMed

    Matos, Rosan Barbosa de; Braga-de-Souza, Suzana; Pitanga, Bruno Pena Seara; Silva, Victor Diógenes Amaral da; Jesus, Erica Etelvina Viana de; Pinheiro, Alexandre Morales; Costa, Maria de Fátima Dias; El-Bacha, Ramon dos Santos; Ribeiro, Cátia Suse de Oliveira; Costa, Silvia Lima

    2014-12-01

    Neospora caninum (Apicomplexa; Sarcocystidae) is a protozoan that causes abortion in cattle, horses, sheep, and dogs as well as neurological and dermatological diseases in dogs. In the central nervous system of dogs infected with N. caninum, cysts were detected that exhibited gliosis and meningitis. Flavonoids are polyphenolic compounds that exhibit antibacterial, antiparasitic, antifungal, and antiviral properties. In this study, we investigated the effects of flavonoids in a well-established in vitro model of N. caninum infection in glial cell cultures. Glial cells were treated individually with 10 different flavonoids, and a subset of cultures was also infected with the NC-1 strain of N. caninum. All of the flavonoids tested induced an increase in the metabolism of glial cells and many of them increased nitrite levels in cultures infected with NC-1 compared to controls and uninfected cultures. Among the flavonoids tested, 3',4'-dihydroxyflavone, 3',4',5,7-tetrahydroxyflavone (luteolin), and 3,3',4',5,6-pentahydroxyflavone (quercetin), also inhibited parasitophorous vacuole formation. Taken together, our findings show that flavonoids modulate glial cell responses, increase NO secretion, and interfere with N. caninum infection and proliferation.

  15. A Conceptual Framework for the Cultural Integration of Cooperative Learning: A Thai Primary Mathematics Education Perspective

    ERIC Educational Resources Information Center

    Park, Ji Yong; Nuntrakune, Tippawan

    2013-01-01

    The Thailand education reform adopted cooperative learning to improve the quality of education. However, it has been reported that the introduction and maintenance of cooperative learning has been difficult and uncertain because of the cultural differences. The study proposed a conceptual framework developed based on making a connection between…

  16. US: A Cultural Mosaic. Teacher Handbook for a Primary-Grade Multidiscipline, Multicultural Program.

    ERIC Educational Resources Information Center

    Martinez, Jimmie; Watters, Arlene

    Activities and objectives for helping elementary school pupils develop a multiethnic and multicultural orientation toward American history and culture are presented in this teacher's guide. The major objective was to develop an interdisciplinary educational program which would influence young children in a positive way as they developed life-long…

  17. ACCUMULATION OF PBDE-47 IN PRIMARY CULTURES OF RAT NEOCORTICAL CELLS.

    EPA Science Inventory

    A number of recent studies have examined the neurotoxic actions of PBDEs using in vitro cell culture models. However, there is little data reporting the final concentration of PBDEs in cells after in vitro exposure to these compounds. To address this issue, the present study exam...

  18. Flavonoids Modulate the Proliferation of Neospora caninum in Glial Cell Primary Cultures

    PubMed Central

    Barbosa de Matos, Rosan; Braga-de-Souza, Suzana; Pena Seara Pitanga, Bruno; Amaral da Silva, Victor Diógenes; Viana de Jesus, Erica Etelvina; Morales Pinheiro, Alexandre; Dias Costa, Maria de Fátima; dos Santos El-Bacha, Ramon; de Oliveira Ribeiro, Cátia Suse

    2014-01-01

    Neospora caninum (Apicomplexa; Sarcocystidae) is a protozoan that causes abortion in cattle, horses, sheep, and dogs as well as neurological and dermatological diseases in dogs. In the central nervous system of dogs infected with N. caninum, cysts were detected that exhibited gliosis and meningitis. Flavonoids are polyphenolic compounds that exhibit antibacterial, antiparasitic, antifungal, and antiviral properties. In this study, we investigated the effects of flavonoids in a well-established in vitro model of N. caninum infection in glial cell cultures. Glial cells were treated individually with 10 different flavonoids, and a subset of cultures was also infected with the NC-1 strain of N. caninum. All of the flavonoids tested induced an increase in the metabolism of glial cells and many of them increased nitrite levels in cultures infected with NC-1 compared to controls and uninfected cultures. Among the flavonoids tested, 3',4'-dihydroxyflavone, 3',4',5,7-tetrahydroxyflavone (luteolin), and 3,3',4',5,6-pentahydroxyflavone (quercetin), also inhibited parasitophorous vacuole formation. Taken together, our findings show that flavonoids modulate glial cell responses, increase NO secretion, and interfere with N. caninum infection and proliferation. PMID:25548412

  19. Variation in pestivirus growth in testicle primary cell culture is more dependent on the individual cell donor than cattle breed.

    PubMed

    Weber, Matheus N; Bauermann, Fernando V; Gómez-Romero, Ninnet; Herring, Andy D; Canal, Cláudio W; Neill, John D; Ridpath, Julia F

    2017-03-01

    The causes of bovine respiratory disease complex (BRDC) are multifactorial and include infection with both viral and bacterial pathogens. Host factors are also involved as different breeds of cattle appear to have different susceptibilities to BRDC. Infection with bovine pestiviruses, including bovine viral diarrhea virus 1 (BVDV1), BVDV2 and 'HoBi'-like viruses, is linked to the development of BRDC. The aim of the present study was to compare the growth of different bovine pestiviruses in primary testicle cell cultures obtained from taurine, indicine and mixed taurine and indicine cattle breeds. Primary cells strains, derived from testicular tissue, were generated from three animals from each breed. Bovine pestivirus strains used were from BVDV-1a, BVDV-1b, BVDV-2a and 'HoBi'-like virus. Growth was compared by determining virus titers after one passage in primary cells. All tests were run in triplicate. Virus titers were determined by endpoint dilution and RT-qPCR. Statistical analysis was performed using one way analysis of variance (ANOVA) followed by the Tukey's Multiple Comparison Test (P˂0.05). Significant differences in virus growth did not correlate with cattle breed. However, significant differences were observed between cells derived from different individuals regardless of breed. Variation in the replication of virus in primary cell strains may reflect a genetic predisposition that favors virus replication.

  20. Kinetics and functional implications of Th1 and Th2 cytokine production following activation of peripheral blood mononuclear cells in primary culture.

    PubMed

    McHugh, S; Deighton, J; Rifkin, I; Ewan, P

    1996-06-01

    The importance of cytokine production in some disease processes is now widely recognized. To investigate temporal relationships between cytokines, we stimulated peripheral blood mononuclear cells (PBMC) in vitro using the T cell mitogen phytohemagglutinin (PHA) and various antigens chosen to induce predominantly Th1 (streptokinase: streptodornase or purified protein derivative) or Th2 (Dermatophagoides pteronyssinus, bee or wasp venom: allergens in sensitive subjects) responses. Cytokine production was measured by sensitive bioassays or enzyme-linked immunosorbent assays. Of the 30 subjects studied, 10 were normal and 20 individuals were allergic to either D. pteronyssinus (n = 10) or bee venom (n = 10) (examined before specific allergen immunotherapy). We examined the temporal profiles of a panel of cytokines produced in primary culture. In PHA-driven cultures, cytokines were found to be sequentially produced in the order interleukin (IL)-2, IL-4, IL-5, IL-3, interferon (IFN)-gamma, IL-10, IL-6, IL-12 and tumor necrosis factor (TNF)-alpha. The response to allergen in allergic patients was predominantly Th2 in nature, with the production of IL-4, IL-5, IL-6 and IL-10, but little or no IFN-gamma. IL-2, IL-3, TNF-alpha and IL-12 were also produced in low amounts. The response of both atopic and normal subjects to recall bacterial antigens was predominantly Th1, with high levels of IFN-gamma, IL-2 and TNF-alpha. The relevance of the order, amount and speed of production, characteristic kinetics (production, consumption, homeostatic regulation) and the cell source of the cytokines are discussed.

  1. Health Imperatives in Primary Schools across Three Countries: Intersections of Class, Culture and Subjectivity

    ERIC Educational Resources Information Center

    Wright, Jan; Burrows, Lisette; Rich, Emma

    2012-01-01

    In this article, we want to focus on the impact of the new health imperatives on young children attending primary schools because the evidence from both our own and others work suggests that younger and younger children are talking in very negative and disturbing ways about themselves and their bodies. We see this in a context where in the name of…

  2. BOLISM OF ARSENITE IN CULTURED PRIMARY HEPATOCYTES FROM SIX MAMMALIAN SPECIES

    EPA Science Inventory

    Inorganic arsenic (iAs) is an environmental toxin and carcinogen. Biomethylation is the major pathway for the metabolism of iAs in many mammalian species, including the human. The liver is considered the primary site for iAs methylation and As (+3 oxidation state) methyltransfera...

  3. The Unreliability of MTT Assay in the Cytotoxic Test of Primary Cultured Glioblastoma Cells.

    PubMed

    Jo, Hwa Yeon; Kim, Yona; Park, Hyung Woo; Moon, Hyo Eun; Bae, Seongtae; Kim, JinWook; Kim, Dong Gyu; Paek, Sun Ha

    2015-09-01

    MTT assay is commonly used to assess the cellular cytotoxicity caused by anticancer drugs in glioblastomas. However, there have been some reports insisting that MTT assay exhibited non-specific intracellular reduction of tetrazolium which led to underestimated results of cytotoxicity. Here, we examine whether or not MTT assay can lead to incorrect information regarding alcohol-induced cytotoxicity on immortalized and primary glioblastoma cells. MTT assay was applied to assess the ethanol-induced cytotoxicity at various ethanol concentrations. The cellular cytotoxicity induced by different doses of ethanol was analyzed and compared through several cytotoxic assays. Ethanol-induced cytotoxicity observed through MTT assay on both cell types was shown to be ethanol dose-dependent below a 3% concentration. However, the cytotoxicity was shown to be markedly underestimated only in primary cells at a 5% concentration. RT-PCR and Western Blot showed increased expressions of pro-apoptotic proteins and decreased expressions of anti-apoptotic proteins in an ethanol dose-dependent manner in both cell types. Furthermore, we present a possible mechanism for the unreliable result of MTT assay. A high concentration of ethanol induces more severe membrane damage and increased intracellular concentration of NADH in primary cells which enhances the nonspecific reduction of tetrazolium salt. Together, our findings demonstrate that the cytotoxicity on primary cells could inaccurately be assessed when detected through MTT assay. Therefore, a careful interpretation is needed when one would analyze the cytotoxic results of MTT assay, and it is suggested that other assays must be accompanied to produce more reliable and accurate cytotoxic results on primary glioblastoma cells.

  4. Enteric oxalate elimination is induced and oxalate is normalized in a mouse model of primary hyperoxaluria following intestinal colonization with Oxalobacter.

    PubMed

    Hatch, Marguerite; Gjymishka, Altin; Salido, Eduardo C; Allison, Milton J; Freel, Robert W

    2011-03-01

    Oxalobacter colonization of rat intestine was previously shown to promote enteric oxalate secretion and elimination, leading to significant reductions in urinary oxalate excretion (Hatch et al. Kidney Int 69: 691-698, 2006). The main goal of the present study, using a mouse model of primary hyperoxaluria type 1 (PH1), was to test the hypothesis that colonization of the mouse gut by Oxalobacter formigenes could enhance enteric oxalate secretion and effectively reduce the hyperoxaluria associated with this genetic disease. Wild-type (WT) mice and mice deficient in liver alanine-glyoxylate aminotransferase (Agxt) exhibiting hyperoxalemia and hyperoxaluria were used in these studies. We compared the unidirectional and net fluxes of oxalate across isolated, short-circuited large intestine of artificially colonized and noncolonized mice. In addition, plasma and urinary oxalate was determined. Our results demonstrate that the cecum and distal colon contribute significantly to enteric oxalate excretion in Oxalobacter-colonized Agxt and WT mice. In colonized Agxt mice, urinary oxalate excretion was reduced 50% (to within the normal range observed for WT mice). Moreover, plasma oxalate concentrations in Agxt mice were also normalized (reduced 50%). Colonization of WT mice was also associated with marked (up to 95%) reductions in urinary oxalate excretion. We conclude that segment-specific effects of Oxalobacter on intestinal oxalate transport in the PH1 mouse model are associated with a normalization of plasma oxalate and urinary oxalate excretion in otherwise hyperoxalemic and hyperoxaluric animals.

  5. HISTONE DEACETYLASE ACTIVITY AND REACTIVE OXYGEN SPECIES CONTENT IN THE TISSUE CULTURE OF Arabidopsis thaliana UNDER NORMAL CONDITIONS AND DEVELOPMENT OF ACUTE OSMOTIC STRESS.

    PubMed

    Jadko, S I

    2015-01-01

    The possible involvement of histone deacetylase (HDAC) in regulation of ROS content in the tissue culture of Arabidopsis thaliana under normal conditions and under development of acute osmotic stress was studied by using inhibition assay with application of trichostatin A (TSA). It was found that in the tissue culture grown under normal conditions a decrease in HDAC activity by means of TSA led to increase of the ROS content. Similar but more pronounced alterations occurred under stress. At the same time an increase in histone acetyltransferase (HAT) activity was also observed. The possible mechanisms of HDAC and HAT participation in regulation of ROS content by changes in expression of genes that are responsible for ROS production and antioxidant activity are discussed.

  6. Cell contact as an independent factor modulating cardiac myocyte hypertrophy and survival in long-term primary culture

    NASA Technical Reports Server (NTRS)

    Clark, W. A.; Decker, M. L.; Behnke-Barclay, M.; Janes, D. M.; Decker, R. S.

    1998-01-01

    . Intercellular contact, communication, and transmission of contractile forces between myocytes appears to play a primary role in remodeling the 2-dimensional cell layer into a parallel alignment of elongated myocytes with highly developed intercalated disk-like junctions. This highly differentiated state is very stable, and cultures which achieve this state exhibit significantly greater longevity than more sparsely plated myocytes. These myocytes typically continue beating, and survive from 6 to more than 12 weeks in culture. When this level of contact and differentiation are not achieved, even among beta-adrenergic stimulated myocytes, contractile activity is not sustained, myofibrils atrophy, there is little or no development of junctional complexes, and the period of myocyte viability is typically no more than 5 weeks in vitro.

  7. Study of Silymarin and Vitamin E Protective Effects on Silver Nanoparticle Toxicity on Mice Liver Primary Cell Culture.

    PubMed

    Faedmaleki, Firouz; Shirazi, Farshad H; Ejtemaeimehr, Shahram; Anjarani, Soghra; Salarian, Amir-Ahmad; Ahmadi Ashtiani, Hamidreza; Rastegar, Hossein

    2016-02-01

    .7 ppm or µg/ml). Then the hepatoprotective effect of silymarin and vitamin E were experimented on silver nanoparticle toxicity on mice liver primary cell culture. The results showed that silymarin at 600 µg/ml and vitamin E at 2500 µmol/l have protective effects on silver nanoparticle toxicity on mice liver primary cell culture. Viability percentage of the primary liver cell of the mouse were exposed to silver nanoparticles at 121.7 ppm and co-treatment of silymarin, and vitamin E is more than viability percentage of the primary liver cell of the mouse were exposed to silver nanoparticles and silymarin or silver nanoparticles and vitamin E.

  8. Quantitative Nuclease Protection Assays (qNPA) as Windows into Chemical-Induced Adaptive Response in Cultures of Primary Human Hepatocytes (Concentration and Time-Response)

    EPA Science Inventory

    Cultures of primary human hepatocytes have been shown to be dynamic in vitro model systems that retain liver-like functionality (e.g. metabolism, transport, induction). We have utilized these culture models to interrogate 309 ToxCast chemicals. The study design characterized both...

  9. Automatically you become a polygamist': 'culture' and 'norms' as resources for normalization and managing accountability in talk about responses to infertility.

    PubMed

    de Kok, B C

    2009-03-01

    In the developing world, infertility is a serious problem. It leads to both psychological and social hardship, in part because childless marriages often result in divorce, men taking another wife or extramarital relationships. Such responses have been attributed to cultural norms that mandate procreation. However, there are theoretical, methodological and moral issues with treating cultural norms as behavioural determinants. They have been insufficiently acknowledged in health research. Therefore, I demonstrate an alternative discursive approach, which examines how people actively mobilize ;culture' or ;norms' in interactions, and the interpersonal functions thereby fulfilled (e.g. blaming or justifying). Analysis is presented of interviews on (responses to) infertility in Malawi. I show how respondents construct polygamy and extramarital affairs as culturally and normatively required, ;automatic' and normal solutions for fertility problems and play down people's accountability for these practices. These accounts and constructions appear to facilitate engagement in affairs and polygamy when people face fertility problems, which seems problematic from a health and gender perspective. Thus, detailed analysis of how people use ;culture' and ;norms' in situ is important because it provides insights into its potentially undesirable consequences. Moreover, such analysis provides a starting point for culturally and gender sensitive interventions, since it highlights people's agency, and creates a space to re-construct and change practices.

  10. Comparison of various detection methods for periodontopathic bacteria: can culture be considered the primary reference standard?

    PubMed Central

    Loesche, W J; Lopatin, D E; Stoll, J; van Poperin, N; Hujoel, P P

    1992-01-01

    The development of diagnostic tests for a periodontal infection raises the issue as to what the appropriate reference standard, or "gold standard," should be for the evaluation of a new test. The present research was initiated to compare the ability of several detection methods, i.e., a serial dilution anaerobic culture and/or microscopic procedure, a DNA probe procedure, and immunological reagents using both an enzyme-linked immunosorbent assay and an indirect immunofluorescence assay to detect Treponema denticola, Porphyromonas gingivalis, Bacteroides forsythus, and Actinobacillus actinomycetemcomitans in subgingival plaque samples taken from 204 periodontally diseased tooth sites. The prevalence of the four monitored species varied as a function of both the species and the detection method. Spirochetes were present in 99% of the plaques, whereas A. actinomycetemcomitans was detected at the lowest frequency. The culture method yielded the lowest prevalence values for the three cultivable species. This raised the question as to which results, those obtained by culture or those obtained by the DNA probes and the immunological reagents, were the most reliable. This issue was addressed by looking at the prevalence profile of the monitored organisms, as determined by all the detection methods. If the species was detected by three or four of the detection methods, then it was considered present, whereas if it was absent by three or four of the detection methods, then it was considered absent. This approach showed the DNA probes and immunological reagents to be significantly superior (P less than 0.05) to the culture approach for the detection of P. gingivalis, A. actinomycetemcomitans, and B. forsythus and to be comparable to the microscopic approach in the detection of T. denticola. PMID:1537912

  11. Comparative effects of contraction and angiotensin II on growth of adult feline cardiocytes in primary culture

    NASA Technical Reports Server (NTRS)

    Wada, H.; Zile, M. R.; Ivester, C. T.; Cooper, G. 4th; McDermott, P. J.

    1996-01-01

    The purposes of this study were 1) to determine whether angiotensin II causes growth of adult feline cardiocytes in long-term culture, 2) to compare the growth effects of angiotensin II with those resulting from electrically stimulated contraction, and 3) to determine whether the anabolic effects of contraction are exerted via the angiotensin type 1 receptor. Adult feline cardiocytes were cultured on laminin-coated trays in a serum-free medium. Cardiocytes were either electrically stimulated to contract (1 Hz, 5-ms pulse duration, alternating polarity) or were nonstimulated and quiescent. Quiescent cells were studied as controls and after treatment with angiotensin II (10(-8) M), losartan (10(-6) M; an angiotensin type 1-receptor antagonist), or angiotensin II plus losartan. Contracting cells were studied in the presence and absence of angiotensin II or losartan. In quiescent cardiocytes, angiotensin II treatment on day 7 significantly increased protein synthesis rates by 22% and protein content per cell by 17%. The effects of angiotensin II were completely blocked by losartan. Electrically stimulated contraction on days 4 and 7 in culture significantly increased protein synthesis rate by 18 and 38% and protein content per cell by 19 and 46%, respectively. Angiotensin II treatment did not further increase protein synthesis rate or protein content in contracting cardiocytes. Furthermore, losartan did not block the anabolic effects of contraction on protein synthesis rates or protein content. In conclusion, angiotensin II can exert a modest anabolic effect on adult feline cardiocytes in culture. In contracting feline cardiocytes, angiotensin II has no effect on growth. Growth caused by electrically stimulated contraction occurs more rapidly and is greater in magnitude than that caused by angiotensin II. Growth of contracting adult feline cardiocytes is not dependent on activation of the angiotensin receptor.

  12. Skeletal muscle satellite cells: background and methods for isolation and analysis in a primary culture system.

    PubMed

    Danoviz, Maria Elena; Yablonka-Reuveni, Zipora

    2012-01-01

    Repair of adult skeletal muscle depends on satellite cells, myogenic stem cells located between the basal lamina and the plasmalemma of the myofiber. Standardized protocols for the isolation and culture of satellite cells are key tools for understanding cell autonomous and extrinsic factors that regulate their performance. Knowledge gained from such studies can contribute important insights to developing strategies for the improvement of muscle repair following trauma and in muscle wasting disorders. This chapter provides an introduction to satellite cell biology and further describes the basic protocol used in our laboratory to isolate and culture satellite cells from adult skeletal muscle. The cell culture conditions detailed herein support proliferation and differentiation of satellite cell progeny and the development of reserve cells, which are thought to reflect the in vivo self-renewal ability of satellite cells. Additionally, this chapter describes our standard immunostaining protocol that allows the characterization of satellite cell progeny by the temporal expression of characteristic transcription factors and structural proteins associated with different stages of myogenic progression. Although emphasis is given here to the isolation and characterization of satellite cells from mouse hindlimb muscles, the protocols are suitable for other muscle types (such as diaphragm and extraocular muscles) and for muscles from other species, including chicken and rat. Altogether, the basic protocols described are straightforward and facilitate the study of diverse aspects of skeletal muscle stem cells.

  13. Attenuation of malonate toxicity in primary mesencephalic cultures using the GABA transport blocker, NO-711.

    PubMed

    Stokes, A H; Bernard, L P; Nicklas, W J; Zeevalk, G D

    2001-04-01

    Cultured rat mesencephalic neurons were used to assess the effects of gamma-aminobutyric acid (GABA) transport blockers on toxicity caused by malonate, a reversible, competitive inhibitor of succinate dehydrogenase. Previous studies utilizing an ex vivo chick retinal preparation have shown that GABA release and cell swelling are early consequences of acute energy impairment and that GABA transport blockers attenuate this toxicity. The present results demonstrate that the nonsubstrate GABA transport blocker, NO-711 (1 nM-1 microM), dose-dependently protected cultured mesencephalic dopamine (DA) and GABA neurons from malonate-induced toxicity. Similar protection was demonstrated with nipecotic acid (1 mM) and SKF89976A (100 nM), substrate and nonsubstrate GABA transport blockers, respectively. These compounds by themselves produced no signs of toxicity, although nipecotic acid caused a long-term decrease in GABA uptake not associated with toxicity. Compounds which decrease intracellular reactive oxygen species (ROS) are protective in this model, but NO-711 did not prevent the rise in intracellular ROS induced by malonate, indicating its protective effects were downstream of ROS production. Supplementation of malonate treated cultures with the GABA(A) agonist, muscimol (10 microM), increased the toxicity toward the DA and GABA neuron populations. Antagonists at the GABA(A) and glycine receptors provided partial protection to both the GABA and DA neurons. These findings suggest that the GABA transporter, GABA(A), and/or glycine channels contribute to cell damage associated with energy impairment in this model.

  14. Airborne culturable fungi in naturally ventilated primary school environments in a subtropical climate

    NASA Astrophysics Data System (ADS)

    Salonen, Heidi; Duchaine, Caroline; Mazaheri, Mandana; Clifford, Sam; Morawska, Lidia

    2015-04-01

    There is currently a lack of reference values for indoor air fungal concentrations to allow for the interpretation of measurement results in subtropical school settings. Analysis of the results of this work established that, in the majority of properly maintained subtropical school buildings, without any major affecting events such as floods or visible mould or moisture contamination, indoor culturable fungi levels were driven by outdoor concentration. The results also allowed us to benchmark the "baseline range" concentrations for total culturable fungi, Penicillium spp., Cladosporium spp. and Aspergillus spp. in such school settings. The measured concentration of total culturable fungi and three individual fungal genera were estimated using Bayesian hierarchical modelling. Pooling of these estimates provided a predictive distribution for concentrations at an unobserved school. The results indicated that "baseline" indoor concentration levels for indoor total fungi, Penicillium spp., Cladosporium spp. and Aspergillus spp. in such school settings were generally ≤1450, ≤680, ≤480 and ≤90 cfu/m3, respectively, and elevated levels would indicate mould damage in building structures. The indoor/outdoor ratio for most classrooms had 95% credible intervals containing 1, indicating that fungi concentrations are generally the same indoors and outdoors at each school. Bayesian fixed effects regression modelling showed that increasing both temperature and humidity resulted in higher levels of fungi concentration.

  15. Primary isolation and serial passage of hepatitis A virus strains in primate cell cultures.

    PubMed

    Binn, L N; Lemon, S M; Marchwicki, R H; Redfield, R R; Gates, N L; Bancroft, W H

    1984-07-01

    Although several primate cell types have been reported to support replication of hepatitis A virus, optimal conditions for the isolation and production of quantities of virus have not been defined. We therefore examined seven different primate cell types for their ability to support replication of primate-passaged and wild-type virus as reflected by intracytoplasmic accumulation of viral antigen (direct immunofluorescence and radioimmunoassay) and propagation of cell culture-adapted virus. Of the cells tested, low-passage African green monkey kidney (AGMK) cells were most sensitive for initial isolation. Viral replication was documented after inoculation of AGMK cells with seven of nine hepatitis A virus antigen-positive fecal specimens (from seven epidemiologically distinct sources). With six inocula, virus was successfully passed in serial cultures. AGMK-adapted virus was readily propagated in continuous AGMK (BS-C-1) cells. The optimal temperature for the growth of virus in BS-C-1 cells was 35 degrees C. Viral release into supernatant fluids was documented in the absence of any cytopathic effect, and infectivity titers in supernatant fluids 21 days after inoculation (50% tissue culture infective does [TCID50], 10(6.0)/ml) equalled or exceeded those in the cell fraction (TCID50, 10(5.5)/ml). Cells maintained in serum-free media readily supported viral growth, with yields of virus (TCID50, 10(6.5)/ml) equal to or greater than those obtained with cells maintained in 2% fetal bovine serum.

  16. Primary isolation and serial passage of hepatitis A virus strains in primate cell cultures.

    PubMed Central

    Binn, L N; Lemon, S M; Marchwicki, R H; Redfield, R R; Gates, N L; Bancroft, W H

    1984-01-01

    Although several primate cell types have been reported to support replication of hepatitis A virus, optimal conditions for the isolation and production of quantities of virus have not been defined. We therefore examined seven different primate cell types for their ability to support replication of primate-passaged and wild-type virus as reflected by intracytoplasmic accumulation of viral antigen (direct immunofluorescence and radioimmunoassay) and propagation of cell culture-adapted virus. Of the cells tested, low-passage African green monkey kidney (AGMK) cells were most sensitive for initial isolation. Viral replication was documented after inoculation of AGMK cells with seven of nine hepatitis A virus antigen-positive fecal specimens (from seven epidemiologically distinct sources). With six inocula, virus was successfully passed in serial cultures. AGMK-adapted virus was readily propagated in continuous AGMK (BS-C-1) cells. The optimal temperature for the growth of virus in BS-C-1 cells was 35 degrees C. Viral release into supernatant fluids was documented in the absence of any cytopathic effect, and infectivity titers in supernatant fluids 21 days after inoculation (50% tissue culture infective does [TCID50], 10(6.0)/ml) equalled or exceeded those in the cell fraction (TCID50, 10(5.5)/ml). Cells maintained in serum-free media readily supported viral growth, with yields of virus (TCID50, 10(6.5)/ml) equal to or greater than those obtained with cells maintained in 2% fetal bovine serum. PMID:6086708

  17. Analysis of central regulatory pathways in p53-deficient primary cultures of malignant fibrous histiocytoma exposed to ifosfamide.

    PubMed

    Schlott, Thilo; Taubert, Helge; Fayyazi, Afshin; Schweyer, Stefan; Bartel, Frank; Korabiowska, Monika; Brinck, Ulrich

    2004-01-01

    Soft tissue sarcomas frequently carry p53 mutations reducing chemotherapeutical response. Especially malignant fibrous histiocytoma (MFH) reveals a reduced ifosfamide (IF) chemosensitivity when compared to other sarcoma entities. This is the first study to analyze MFH cells for the effects of IF on the expression of the pathways P16-CDK4-Rb and P14ARF-MDM2-P73 regulating cell cycle. The aim was to identify candidate genes possibly involved in the anti-apoptotic response of p53-deficient MFH cells during chemotherapy. PCR, real-time RT-PCR and confocal laser scanning microscopy were applied on primary cultures of MFH cells containing defective p53 genes. The cultures were treated with different concentrations of IF. A non-treated MFH culture served as negative control. A threshold concentration of IF (100 microM) was determined sparing the majority of the cells (99%), whereas higher IF quantities caused complete apoptosis. Data collected over a period of 48 h showed that the MFH cells surviving 100 microM IF overexpressed the kinase gene CDK4 and oncogene MDM2 by a factor of 63. A similar strong increase was observed at the protein level for both proteins. In contrast, the other proteins analyzed were not detectable. Additionally, the MFH cells induced complex patterns of MDM2 mRNA splicing and a